Pyrin gene and mutants thereof, which cause familial Mediterranean fever

ABSTRACT

The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

This is a 35 U.S.C. §371 national phase application of, and claims priority to, international application PCT/US98/17255, filed Aug. 20, 1998, which claims priority, under 35 U.S.C. §119(e), to provisional application U.S. Ser. No. 60/056,217, filed Aug. 21, 1997, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel genomic DNA sequence (MEFV) encoding a protein (pyrin) associated with familial Mediterranean fever (FMF). More specifically, the invention relates to the isolation and characterization of MEFV, and the correlation of mutations in MEFV with FMF disease.

2. Background of the Invention

Familial Mediterranean Fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever, serosal inflammation and abdominal pain. This inflammatory disorder is episodic, with self-limited bouts of fever accompanied by unexplained arthritis, sterile peritonitis, pleurisy and/or skin rash. Patients often develop progressive systemic amyloidosis from the deposition of the acute phase reactant serum amyloid A (SAA). In some patients, progressive systemic amyloidosis can lead to kidney failure and death. The factors which incite an episode are unclear.

FMF is observed primarily in individuals of non-Ashkenazi Jewish, Armenian, Arab and Turkish background. Although rare in the United States, incidence of FMF in Middle Eastern populations can be as high as 1:7 in Armenian populations and 1:5 in non-Ashkenazi Jewish populations.

FMF attacks are characterized by a massive influx of polymorphonuclear leukocytes (PMNs) into the affected anatomic compartment. At the biochemical level, patients have been reported to have abnormal levels of C5a inhibitor (Matzner and Brzezinski, “C5a-inhibitor deficiency in peritoneal fluids from patients with familial Mediterranean fever,” N. Engl. J. Med., 311:287-290 (1984)), neutrophil-stimulatory dihydroxy fatty acids (Aisen et al, “Circulating hydroxy fatty acids in familial Mediterranean fever,” Proc. Natl. Acad. Sci. USA, 2:1232-1236 (1985)), and dopamine β-hydroxylase (Barakat et al, “Plasma dopamine beta-hyroxylase: rapid diagnostic test for recurrent hereditary polyserositis,” Lancet, 2:1280-1283 (1988)). Although linkage studies have placed the gene causing FMF (designated MEFV) on chromosome 16p (Pras et al., “Mapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16,” N. Engl. J. Med., 326:1509-1513 (1992); Shohat et al., “The gene for familial Mediterranean fever in both Armenians and non-Ashkenazi Jews is linked to the α-globin complex on 16p: evidence for locus homogeneity,” Am. J. Hum. Genet., 51:1349-1354 (1992); Pras et al, “The gene causing familial Mediterranean fever maps to the short arm of chromosome 16 in Druze and Moslem Arab families,” Hum. Genet., 94:576-577(1994); French FMF Consortium, “Localization of the familial Mediterranean fever gene (FMF) to a 250 kb-interval in non-Ashkenazi Jewish founder haplotypes,” Am. J. Hum. Genet., 59:603-612(1996)), the genetic basis of FMF has not previously been identified.

Current treatment regimens for FMF include daily oral administration of colchicine. Although colchicine has been shown to cause near complete remission in about 75% of FMF patients and prevent amyloidosis, colchicine is not effective in all patients. Therefore, there is a need for new treatments for colchicine-resistant patients.

Additionally, there is a need for an accurate diagnostic test for FMF. Patients having FMF in countries where the disease is less prevalent often experience years of attacks and several exploratory surgeries before the correct diagnosis is made.

SUMMARY OF THE INVENTION

The invention provides a novel genomic nucleic acid sequence (MEFV) (SEQ ID NO:1), shown in FIG. 1, encoding the protein pyrin which is associated with familial Mediterranean fever (FMF). The corresponding cDNA sequence (v75-1) (SEQ ID NO:2) and encoded amino acid sequence (SEQ ID NO:3) are shown in FIG. 2. The invention is also directed towards fragments of the DNA sequence that are useful, for example, as hybridization probes for diagnostic assays or oligonucleotides for PCR priming. Additionally, the invention is directed towards the corresponding sequence for the RNA transcript and fragments thereof.

Another aspect of the invention provides the amino acid sequence for a protein associated with FMF. This protein is called pyrin, to connote its relationship to fever. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. These proteins are useful, for example, as antigens to produce specific anti-pyrin antibodies to be used as agents in diagnostic assays. Alternatively, the protein may be used in therapeutic compositions.

Mutations in pyrin result in FMF. Therefore, the invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain (SEQ ID NO:5) at the C-terminal of the protein. These mutants include M680I, M694V, K695R and V726A, each of which is associated with FMF.

Additionally, the invention includes methods for diagnosing a patient at risk for having FMF using the nucleic acid and/or amino acid sequences of the invention. Such methods include, for example, hybridization techniques using nucleic acid sequences, PCR-amplification of MEFV, and immunoassays using anti-pyrin antibodies to identify mutations is MEFV or pyrin which are indicative of FMF.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic nucleic acid sequence for the gene associated with FMF;

FIG. 2 shows a cDNA sequence and deduced amino acid sequence corresponding to the gene associated with FMF;

FIG. 3 is a schematic representation of MEFV on chromosome 16p13.3;

FIG. 4 show the expression profile of V75-1;

FIG. 5 shows the DNA sequences of the M6801, M694V and V726A mutants; and

FIG. 6 shows the alignment of multiple protein sequences with the C-terminal end of human pyrin.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the nucleic acid sequence encoding a protein associated with familial Mediterranean fever (FMF). The genomic DNA sequence is designated MEFV. The corresponding cDNA sequence is designated as v75-1. The encoded protein is called pyrin, to connote its relationship to fever. The inventors have also discovered mutations in MEFV which are associated with FMF.

It is believed that pyrin is a nuclear factor that controls the inflammatory response in differentiated polymorphonuclear leukocytes (PMNs). In particular, pyrin is believed to be a negative autoregulatory molecule in PMNs. Knowledge of the genetic basis of FMF enables the production of diagnostic assays for FMF and treatments for FMF and other inflammatory diseases which are characterized by accumulation of PMNs, for example, acute infectious disease such as those caused by bacterial infection (e.g., Pneumococcal pneumonia), autoimmune diseases such as Sweets Syndrome or Behcet's disease, chronic arthritis, and the like.

The Nucleic Acid Sequence (MEFV)

The inventors have discovered the nucleic acid sequence for the gene associated with FMF. The nucleic acid sequence is found on chromosome 16p. Specifically, MEFV is located at 16p13.3 between the polycystic kidney disease gene (PKD1) and the tuberous sclerosis gene (TSC2) on the telomeric end, and the CREB-binding protein gene (CREBBP) on the centromeric end (see FIG. 3).

The genomic DNA sequence encoding pyrin (MEFV) (SEQ ID NO:1) is shown in FIG. 1. The start methionine and stop codon are boxed, while the exons are underlined. The cDNA sequence (v75-1) (SEQ ID NO:2) is shown in FIG. 2. In FIG. 2, the initial methionine and Kozak consensus sequences are underlined. The first boxed segment is a bZIP transcription factor basic domain. The second boxed segment is a Robbins/Dingwall consensus nuclear targeting signal. The segment indicated by +'s is a potential B-box zinc finger domain. The double-boxed region encloses a sequence which encodes a rfp, or B30.2, domain (SEQ ID NO:4). Within the double boxed region (the rfp or B30.2 domain), the nucleic acids encoding three FMF-associated mutations are double-underlined. Sites of synonymous single nucleotide polymorphisms are represented by the cents symbol “¢” above the sequence.

Although there is an excellent Kozak consensus sequence (Kozak, “Interpreting cDNA sequences: some insights from studies on translation,” Mamm. Genome, 7:563-574 (1996)) at the initial methionine (accATGG), the reading frame remains open in the cDNA upstream. Because there are no splice-acceptor consensus sequences or in-frame methionines with good Kozak sequences before the first stop upstream in the genomic DNA, the initial methionine remains the most likely starting methionine.

The RNA Transcript

The estimated transcript size from the nucleic acid sequence shown in FIG. 2 is about 3503 nucleotides. The transcript size determined by Northern blotting is 3.7 kb. (See Example 4). The fact that the transcript size estimated from the sequence shown in FIG. 2 approximates the size of the transcript found in experimental procedures further indicates that the sequence shown in FIG. 2 is the full-length cDNA sequence.

The Encoded Protein

The inventors have also discovered the amino acid sequence for the protein associated with FMF (pyrin). Pyrin is predicted to be 781 amino acids in length and very positively charged. The pI is predicted to be greater than 8 (pI>8), in part due to the fact that lysine and arginine residues make up 13% of the amino acid composition.

The predicted amino acid sequence for pyrin (SEQ ID NO:3) is shown in FIG. 2. The boxed segment from amino acid 266 to 280 is a bZIP transcription factor basic domain. The boxed segment from amino acid 420 to 437 is a Robbins/Dingwall consensus nuclear targeting signal. The segment indicated by +'s between residues 375 and 407 is a potential B-box zinc finger domain. The region double-boxed from residue 577 to 757 is a rfp, or B30.2, domain (SEQ ID NO:5). The rfp (B30.2) domain is conserved (sequence identity 40-60%) in molecules as diverse as butyrophilin (a milk protein with probably receptor function; Jack and Mather, “Cloning and molecular analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation,” J. Biol. Chem., 265:14481-14486 (1990)), A33 (a factor that binds polytene chromosomes in the newt; Bellini et al., “A putative zinc-binding protein on lampbrush chromosome loops,” EMBO J., 12:107-114 (1993)), and xnf7 (a factor that binds mitotic chromosomes in the frog; Reddy et al., “The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis,” Dev. Biol. 148:107-116 (1991)) and, by an analysis with the SEG algorithm (Wootton, “Non-globular domains in protein sequences: automated segmentation using complexity measures,” Comput. Chem., 18:269-285 (1994)), most likely assumes a globular conformation. Within the double boxed region (the rfp or B30.2 domain), three of the amino acids that have been found mutated in FMF patients are double-underlined.

Expression

Pyrin is predominantly expressed in mature granulocytes and/or serosal cells. As shown in the Northern blots in FIG. 4, high levels of pyrin are expressed in peripheral blood leukocytes (granulocytes), but not in lymph nodes, bone marrow, monocytes, lymphocytes, spleen or thymus (See FIG. 4). Because granulocytes accumulate in tissues experiencing inflammation during a FMF episode, expression of pyrin in granulocytes is consistent with the clinical phenotype for FMF.

The restriction of pyrin to granulocytes, its apparent localization in the nucleus, and the phenotype associated with mutations tends to indicate that pyrin is a nuclear factor that controls the inflammatory response in differentiated PMNs. Additionally, the inventors found that pyrin shares homology with a number of molecules implicated in inflammation, such as rpt-1 (a known downregulator of inflammation). In view of the fact that FMF is a disease of excessive inflammation, and that pyrin shares homology to a known downregulator of inflammation, pyrin is believed to be a negative autoregulatory molecule in PMNs.

Homologies

Pyrin shares homology with a number of molecules implicated in inflammation including 52 kd Ro/SS A ribonucleoprotein (patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome frequently make autoantibodies against this ribonucleoprotein); Staf-50 (an interferon-inducible transcriptional regulator; Tissot and Mechti, “Molecular cloning of a new interferon-induced factor that represses human immunodeficiency virus type 1 long terminal repeat expression,” J. Biol. Chem., 270:14891-14898 (1995)); and rpt-1 (a mouse downregulator of IL-2; Patarca et al., “rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1,” Proc. Natl. Acad. Sci. USA, 85:2733-2737 (1988)).

The homology between pyrin and rpt-1 is found in a domain extending from residues 385-550 on pyrin. Pyrin shows particularly high homology to many proteins, including 50 kdRo/SS A and Staf-50, at the C-terminal end, the rfp (B30.2) domain. FIG. 6 shows the alignment of the C-terminal end of human pyrin with multiple sequences having statistical similarity as assessed by BLAST (Altschul et al., supra). Search cutoffs used to identify homologs were a Karlin-Altschul score of two aligned sequences ≧70 with a probability ≦10⁻³. At each position, residues occurring in a majority of the sequences are shown in inverse type. The numbering scheme at the top of the figure is based on the sequence of pyrin.

The B-box zinc finger and rfp (B30.2) domain combination observed in pyrin is also seen in 52 kd Ro/SS A and ret finger protein. The spacing between the B-box zinc finger and the rfp (B30.2) domain is highly conserved, suggesting that precise orientation of the two domains with respect to one another may be required for function.

Mutants

The inventors have also discovered missense mutations that are found in individuals affected with FMF, but not found in any of a large panel of normal control chromosomes. The missense mutations are clustered within about 40 to 50 amino acids (including residues 680 through 726) in the highly conserved rfp (B30.2) globular domain. It is believed that the mutations affect the secondary structure of this domain and result in a structural change that prevents the normal pyrin-mediated negative feedback loop.

A first mutation associated with FMF is a G C transversion at nucleotide 2040 which results in the substitution of isoleucine for methionine (M680I). A second mutation is an A G transition at nucleotide 2080 which results in the substitution of valine for methionine (M694V). A third mutation is a T C transition at nucleotide 2177 which results in the substitution of alanine for valine (V726A). Additionally, the inventors have discovered a fourth mutation at position 695 which results in the substitution of Arginine for Lysine (K695R).

It is believed that phenotypic variation in FMF may be attributable to the differences between mutations. For example, the M694V mutation is very common in populations with the highest incidence of systemic amyloidosis (especially North African Jews). On the other hand, V726A is seen in populations in which amyloid is less common (Iraqi and Ashkenazi Jews, Druze and Armenians).

FIG. 5 shows DNA sequence electropherograms, produced by amplifying exon 10 genomic DNA and sequencing, which demonstrate the M680I, M694V, and V726A substitutions. For each mutation, individuals who are homozygous for the normal allele are shown at the top, heterozygotes between the normal and mutant allele are shown in the middle, and homozygotes for the mutation are shown at the bottom.

None of these mutations result in a truncated protein. This is consistent with the periodic nature of the inflammatory attacks in FMF. Other diseases with periodic episodes are associated with a protein that functions adequately at steady state, but decompensates under stress, such as sickle cell anemia (Weatherall et al., “The hemoglobinopathies,” In The Metabolic and Molecular Bases of Inherited Disease, Scriver et al, eds., New York, McGraw-Hill, pp. 3417-3484 (1995) and hyperkalemic periodic paralysis (Ptacek et al., “Identification of a mutation in the gene causing hyperkalemic periodic paralysis,” Cell, 67:1021-1027 (1991)).

Diagnostic Methods

The sequences provided by this invention can be used in methods for diagnosis of risk for developing FMF. As used herein, an individual is “at risk” for developing FMF when the individual has a mutant MEFV nucleic acid sequence which results in expression of mutant pyrin, particularly where the amino acid mutation occurs in the highly conserved rfp (B30.2) C-terminal domain. Mutations include substitutions of one nucleic acid with a different nucleic acid. In contrast, a patient having wild type MEFV nucleic acid sequence expressing wild type pyrin is not at risk for developing FMF. As used herein, “wild type” refers to a dominant genotype which naturally occurs in the normal population (i.e., members of the population not afflicted with familial Mediterranean fever). Thus, methods for identifying an individual's specific nucleic acid or amino acid sequence are useful for determining risk of FMF. Specifically, a method for determining whether an individual's nucleic acid sequence encodes a wild type or mutant pyrin is useful in determining whether the individual is at risk for developing FMF.

Many methods for analysis of an individuals nucleic acid or amino acid sequences are known to those of skill in the art, and include, for example, direct sequencing, ARMS (amplification refractory mutation system), restriction endonuclease assays, oligonucleotide hybridization techniques, and immunoassays. While some commonly used procedures are exemplified below, the inventors are aware that other methods are available and include them within the scope of their invention.

Southern Blot Techniques

In Southern blot analysis, DNA is obtained from an individual and then separated by gel electrophoresis. Following electrophoresis, the double stranded DNA is converted to single stranded DNA, for example, by soaking the gel in NaOH. The DNA is then transferred to a sheet of nitrocellulose. The DNA is then contacted with a labeled probe. For example, labeled probe can be applied to the nitrocellulose after it dries. As used herein, a “probe” is a nucleic acid sequence that is complementary to the sequence of interest. The probe can be either a DNA sequence or an RNA sequence. Preferably the probe is about 8 to 16 nucleotides in length. A radioactive label, such as ³²P is an example of a suitable label. Other suitable labels include fluorophores or an enzyme which catalyzes a color producing reaction (e.g., horse radish peroxidase). Because the probe has complementary sequence to the DNA sequence of interest, it will hybridize to the specific DNA sequence. As used herein, “hybridize” means that the probe will form a double-stranded molecule with the specific DNA sequence by complementary base pairing under conditions of high stringency (e.g., 65° C.; 0.1×SSC; Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (1989)). After the probe is allowed to hybridize to the DNA, excess probe is washed away. The hybridized DNA is easily visualized from the labeled probe using known techniques. Hybridization of the probe indicates that the sample DNA contains a sequence that is complementary to the labeled probe. In a preferred method, hybridization probes are designed from the MEFV nucleic acid sequences, and particularly, from the C-terminal MEFV sequence encoding the rfp (B30.2) globular domain.

It is often desirable to amplify the sample DNA for more efficient analysis. Polymerase chain reaction (PCR) can be used to amplify the DNA. PCR is a technique that is well known to one of skill in the art. An exemplary method includes developing oligonucleotide primers that hybridize to opposite strands of DNA flanking the MEFV gene. As used herein, a “primer” is a short nucleotide sequence which is complementary to a DNA sequence flanking the DNA sequence of interest. Preferably the primer is about 15 to 20 nucleotides in length. The specific fragment defined by the primers exponentially accumulates by repeated cycles of denaturation, oligonucleotide primer annealing and primer extension. In a preferred embodiment, the PCR primers amplify the region encoding the rfp (B30.2) globular domain. The amplified domain can then be analyzed by hybridization or screening techniques.

For example, oligonucleotide primers are developed to amplify MEFV, the rfp (B30.2) domain, or a fragment thereof, such as the preferred 40 to 50 amino acid fragment of the rfp (B30.2) domain discussed above. Suitable oligonucleotide primers, such as “Exon 10A Forward and Reverse”, “Exon 10B Forward and Reverse”, and “Exon 10B Forward and Exon 10A Reverse”, are shown in Example 1.

Northern Blot Techniques

The presence of a wild type or mutant RNA transcript may be determined by Northern Blot Techniques, following a procedure similar to that outlined for the Southern Blot Technique.

Western Blot Techniques

The presence of a wild type or mutant protein from the highly conserved C-terminal rfp (B30.2) region can be detected by immunoassay, for example by Western Blot Techniques. In this procedure, a tissue sample is obtained from an individual and separated by gel electrophoresis. Following electrophoresis, the proteins are then transferred to nitrocellulose. The proteins are then contacted with a labeled probe, for example, by applying the labeled probe to the nitrocellulose after it is dried. Suitable probes include labeled anti-pyrin antibodies, preferably those antibodies specific for an epitope in the highly conserved C-terminal rfp (B30.2) domain. Exemplary labels include radioactive isotopes, enzymes, fluorophores and chromophores. Because it is believed that mutants in the highly conserved C-terminal domain alter the secondary structure of the domain, an antibody specific for the wild-type protein should not bind to or recognize a protein having a mutation in this highly conserved region. Conversely, an antibody specific for a mutant protein does not recognize or bind to the wild type. After excess antibody is rinsed away, the presence of the specific protein/antibody complex is easily determined by known methods, for example by development of the label attached to the anti-pyrin antibody, or by the use of secondary antibodies.

Sequencing Techniques

Alternately, DNA, RNA or protein obtained from an individual can be sequenced by known methods, and compared to the wild type sequence. Mutations recognized in the sequence, particularly, in the rfp (B30.2) domain indicate risk for developing FMF.

ARMS

ARMS (amplification refractory mutation system) is a PCR based technique in which an oligonucleotide primer that is complementary to either a normal allele or mutant allele is used to amplify a DNA sample. In one variation of this method, a pair of primers is used in which one primer is complementary to a known mutant sequence. If the DNA sample is amplified, the presence of the mutant sequence is confirmed. Lack of amplification indicates that the mutant sequence is not present. In a different variation, the primers are complementary to wild type sequences. Amplification of the DNA sample, indicated that the DNA has the wild type sequence complementary to the primers. If no amplification occurs, the DNA likely contains a mutation at the sequence where hybridization should have occurred. A description of ARMS can be found in Current Protocols in Human Genetics, Chapter 9.8, John Wiley & Sons, ed by Dracopoli et al. (1995).

Restriction Endonuclease Assays

Restriction endonuclease assays can also be used to screen a DNA sample for mutants, such assays are used by Pras et al., “Mutations in the SLC3A1 transporter gene in Cystinuria,” Am. J. Hum. Genet., 56:1297-1303 (1995). Briefly, a DNA sample is amplified and then exposed to restriction endonucleases that will or will not cleave the DNA depending on whether or not a mutation is present. After cleavage, the size of restriction fragments are observed to determine whether or not cleavage occurred.

Oligonucleotide Hybridization Techniques

Hybridization techniques, such as dot blots, are known to one of skill in the art and can be used to determine whether a DNA sample contains a specific sequence. In a dot blot, a DNA sample is denatured and exposed to a labeled probe which is complementary for a wild type sequence or a mutant sequence. Hybridization of a probe that is complementary to the wild type sequence (a “wild type probe”) indicates that the wild type sequence is present. If the wild type probe does not hybridize to the DNA in the sample, the wild type sequence is not present. In a variation of this technique a probe that is complementary to a know mutant sequence can be used. A discussion of allele specific oligonucleotide testing can be found in Current Protocols in Human Genetics, Chapter 9.4, supra.

Immunological Assays

An immunological assay, such as an Enzyme Linked Immunoassay (ELISA), can be used as a diagnostic tool to determine whether or not an individual is at risk for developing FMF. One of skill in the art is familiar with the procedure for performing an ELISA. Briefly, antibodies are generated against native or mutant pyrin. This can be accomplished by administering a native or mutant protein to an animal, such as a rabbit. The anti-pyrin antibodies are purified and screened to determine specificity. In one representative example of an immunoassay, wells of a microtiter plate are coated with the specific anti-pyrin antibodies. An aliquot of a sample from a patient to be analyzed for pyrin is added in serial dilution to each antibody coated well. The sample is then contacted with labeled anti-pyrin antibodies. For example, labeled anti-pyrin antibodies, such as biotinylated anti-pyrin antibodies, can be added to the microtiter plate as secondary antibodies. Detection of the label is correlated with the specific pyrin antigen assayed. Other examples of suitable secondary antibody labels include radioactive isotopes, enzymes, fluorophores or chromophores. The presence of bound labeled (biotinylated) antibody is determined by the interaction of the biotin with avidin coupled to peroxidase. The activity of the bound peroxidase is easily determined by known methods.

Production of Pyrin

The nucleic acid sequence encoding wild type or mutant pyrin can be used to produce pyrin in cells transformed with the sequence. For example, cells can be transformed by known techniques with an expression vector containing v75-1 cDNA sequence operably linked to a functional promoter. Expression of pyrin in transformed cells is useful in vitro to produce large amounts of the protein. Expression in vivo is useful to provide the protein to pyrin-deficient cells. Examples of suitable host cells include animal cells such as bacterial or yeast cells, for example, E. coli. Additionally, mammalian cells, such as Chinese hamster ovary (CHO) cells can be used. Human cells, such as SW480 colorectal adenocarcinoma can also be used as host cells.

Due to degeneracy of the genetic code, most amino acids are encoded by more than one codon. Therefore, applicants recognize, and include within the scope of the invention, variations of the sequence shown in SEQ ID NO: 1. For example, codons in a DNA sequence encoding pyrin can be modified to reflect the optimal codon frequencies observed in a specific host. Rare codons having a frequency of less than about 20% in known sequences of the desired host are preferably replaced with higher frequency codons.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences including spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and other well characterized sequences which may be deleterious to gene expression. The G-C content of a sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. The genomic sequence might additionally be modified by the removal of introns.

Transgenic Animals

The nucleic acid sequences encoding pyrin, both wild-type and mutant, provided in this application are useful for the development of transgenic animals expressing pyrin. Such transgenic animals are used, for example, to screen compounds for treating FMF or inflammation.

Useful variations of a transgenic animal are “knock out” or “knock in” animals. In a “knock out” animal, a known gene sequence, such as the sequence encoding pyrin, is deleted from the animal's genome. Experiments can be performed on the animal to determine what effect the absence of the gene has on the animal. In a “knock in” experiment, the wild type gene is deleted and a mutant version or a gene from another organism is inserted therefore. Experiments can be performed on the animal to determine the effects of this transition.

Kits

The invention is also directed towards a kit for diagnosing risk of FMF. A suitable diagnostic kit includes a nucleic acid sequence encoding wild-type pyrin and at least one nucleic acid sequence encoding mutant pyrin. An alternative kit includes an anti-pyrin antibody which binds to wild-type pyrin and at least one anti-pyrin antibody which binds to mutant pyrin. A kit also preferably contains at least one pair of amplification primers capable of amplifying a nucleic acid sequence encoding pyrin. Preferably, the primers amplify a nucleic acid sequence encoding a rfp (B30.2) domain of pyrin.

The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

EXAMPLES

The DNA samples used in the following examples were extracted from whole blood or from Epstein-Barr virus-transformed lymphocytes by standard techniques. The DNA was obtained from forty-four families of non-Ashkenazi Jewish descent (18 Moroccan, 14 Libyan, 5 Tunisian, 2 Egyptian and 5 Iraqi) and 5 Arab/Druze families (identified and sampled at the Chaim Sheba Medical Center in Tel-Hashomer, Israel). Additionally, twelve Armenian families were recruited from Cedars-Sinai Medical Center in Los Angeles. One Ashkenazi/Iraqi Jewish family was also studied.

The diagnosis of FMF in all families was according to established clinical criteria (Sohar et al., “Familial Mediterranean fever: a survey of 470 cases and review of the literature,” Am. J. Med., 43:227-253 (1967)).

Example 1 Positional Cloning

A positional cloning approach was used to clone a new cDNA (v75-1) from the FMF candidate region on chromosome 16p13.3. Mutational analysis indicates the v75-1 is the gene (designated MEFV) expressing pyrin, mutations of which are associated with FMF disorder.

Publicly available polymorphic markers (discussed below) were used to narrow the candidate region on chromosome 16p to an approximately 1 Mb interval between D16S94 and D16S2622 (Sood et al., “Construction of a 1-Mb restriction mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p13.3,” Genomics, 42:83-95 (1997)) lying between the polycystic kidney disease (PKD1) and tuberous sclerosis (TSC2) genes on the telomeric end, and the CREB-binding protein (CREBBP) gene on the centromeric end (see FIG. 3). Because physical maps constructed around these genes did not extend into the MEFV region, a contig was constructed which spanned the candidate region.

Attempts to construct a mega YAC (yeast artificial chromosome) contig spanning the MEFV candidate region were unsuccessful due to the instability of YAC clones from this region of chromosome 16. Instead, a cosmid map was assembled by iterative screening of a flow sited chromosome 16 specific cosmid library. D16S246 was the telomeric starting point of the chromosomal walk. Identification of recombinants at D16S2622 enabled us to use this microsatellite marker as the centromeric boundary (Sood et al., 1997, supra).

Observed recombinations of microsatellite markers in a panel of 61 families defined a critical region of 285 kb (D16S468-D16S3376).

By analysis of the genomic sequence from this region, two new microsatellites, D16S3404 and D16S3405 (FIG. 3B), were found in the center of the D16S3082-D16S3373 interval. In one non-Ashkenazi Jewish family, evidence of a historical recombination event between D16S3404 and D16S3405 in the highly conserved non-Ashkenazi Jewish haplotype (designated haplotype A) was observed. Therefore, the region telomeric of D16S3405 (and 4 candidate genes encoded therein) were excluded from further consideration. The discovery of the two new microsatellites and the historical recombination event further refined of the candidate interval to the centromeric-most 115 kb.

A combined strategy of exon amplification, direct cDNA selection, and single-pass sequencing led to the isolation of 9 full length cDNA clones. The furthest centromeric cDNA clone, v75-1, was isolated by solution hybridization of a leukocyte cDNA library with biotinylated oligonucleotide probes derived from two exons trapped from PAC 273L24.

Exon Trapping

PAC (P1 artificial chromosome) clone 273L24 (Genome Systems; St. Louis) includes the centromeric-most 115 kb. Therefore, exon trapping was performed on PAC clone 273L24. Exon trapping was performed essentially as described by Buckler et al., “Exon amplification: a strategy to isolate mammalian genes based on RNA splicing,” Proc. Natl. Acad. Sci. USA, 88:4005-4009 (1991). Essentially, PAC clone 273L24 was partially digested with Sau 3AI (commercially available, for example, from New England Biolabs). The reaction products were size fractionated by agarose gel electrophoresis and DNA fragments 2 kb and larger were isolated from the gel. Fifty ng of partially digested DNA was ligated with 10 ng of exon trapping vector pSPL3 (Exon Trapping System; Life Technologies, Gaithersburg, Md.) that had been previously cleaved with Bam HI (commercially available) and dephosphorylated with calf intestinal alkaline phosphatase (Promega, Madison, Wis.). Ligation products were electroporated into E. coli DH12B (Life Technologies, Gaithersburg, Md.) The electroporated cells were cultured en mass in LB broth with 200 mg/ml ampicillin for 16 hours at 37° C. with shaking.

DNA prepared from the culture was used to transfect COS-7 cells (ATCC 30-2002) using lipofectACE reagent (Life Technologies, Gaithersburg, Md.). Total RNA was isolated from transfected COS-7 cells with Trizol reagent (Life Technologies) followed by ethanol precipitation.

First strand cDNAs of transcription products from pSPL3 were primed with the oligonucleotide SA2 (Exon Trapping System; Life Technologies, Gaithersburg, Md.). Specific amplification of trapped exons was as follows: PCR primed with oligonucleotides SA2 and SD6 (Exon Trapping System; Life Technologies, Gaithersburg, Md.) was performed, followed by digestion of the PCR products with Bst XI (commercially available).

A second PCR reaction using the digestion products was primed with oligonucleotides dUSD2 and dUSA4 (Exon Trapping System; Life Technologies, Gaithersburg, Md.). The resulting DNA fragments were cloned into pAMP10 vector (Exon Trapping System; Life Technologies, Gaithersburg, Md.) and sequenced. Two hundred clones were sequenced and 20 independent exons were identified by visual inspection and hybridization to DNA fragments from the FMF critical region, with several exons identified more than one time.

Oligonucleotides for Exon Amplification

Oligonucleotides used to amplify pyrin exons were as follows (all oligo sequences are given 5′ to 3′):

Exon 1 forward, AAC CTG CCT TTT CTT GCT CA; (SEQ ID NO:6)

Exon 1 reverse, CAC TCA GCA CTG GAT GAG GA; (SEQ ID NO:7)

Exon 2A forward, ATC ATT TTG CAT CTG GTT GTC CTT CC; (SEQ ID NO:8)

Exon 2A reverse, TCC CCT GTA GAA ATG GTG ACC TCA AG; (SEQ ID NO:9)

Exon 2B forward, GGC CGG GAG GGG GCT GTC GAG GAA GC; (SEQ ID NO:10)

Exon 2B reverse, TCG TGC CCG GCC AGC CAT TCT TTC TC; (SEQ ID NO:11)

Exon 3 forward, TGA GAA CTC GCA CAT CTC AGG C; (SEQ ID NO:12)

Exon 3 reverse, AAG GCC CAG TGT GTC CAA GTG C; (SEQ ID NO:13)

Exon 4 forward, TTG GCA CCA GCT AAA GAT GGC; (SEQ ID NO:14)

Exon 4 reverse, TCT CCC TCT ACA GGG ATG AGC; (SEQ ID NO:15)

Exon 5 forward, TAT CGC CTC CTG CTC TGG AAT C; (SEQ ID NO:16)

Exon 5 reverse, CAC TGT GGG TCA CCA AGA CCA AG; (SEQ ID NO:17)

Exon 6 forward, TCC AGG AGC CCA GAA GTA GAG; (SEQ ID NO:18)

Exon 6 reverse, TTC TCC CTA TCA AAT CCA GAG; (SEQ ID NO:19)

Exon 7 forward, AGA ATG TAG TTC ATT TCC AGC; (SEQ ID NO:20)

Exon 7 reverse, CAT TTC TGA ACG CAG GGT TT; (SEQ ID NO:21)

Exon 8/9 forward, ACC TAA CTC CAG CTT CTC TCT GC; (SEQ ID NO:22)

Exon 8/9 reverse, AGT TCT TCT GGA ACG TGG TAG; (SEQ ID NO:23)

Exon 10A forward, CCA GAA GAA CTA CCC TGT CCC; (SEQ ID NO:24)

Exon 10A reverse, AGA GCA GCT GGC GAA TGT AT; (SEQ ID NO:25)

Exon 10B forward, GAG GTG GAG GTT GGA GAC AA; (SEQ ID NO:26)

Exon 10B reverse, TCC TCC TCT GAA ATC CAT GG. (SEQ ID NO:27).

Direct cDNA selection

Direct cDNA selection was used to isolate 2 full-length cDNA clones (Parimoo et al., “cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments,” Proc. Natl. Acad. Sci. USA, 88:9623-9627 (1991). Cosmids, BAC (bacterial artificial chromosome) and P1 clones in the FMF candidate region were biotinylated using BioPrime (Life Technologies, Gaithersburg, Md.). cDNAs were prepared from combined mRNA from fetal brain, fetal liver, and human lymph node by reverse transcription and ligation of an EcoRI/NotI adaptor to second strand cDNAs.

cDNAs were directly hybridized to biotinylated templates which were recovered using streptavidin-labeled magnetic beads. Conditions for blocking, hybridization, binding and elution of cDNAs from magnetic beads (Dynal) were as described by Parimoo et al., supra. After two rounds of selection, eluted cDNAs were amplified with CUA-tailed EcoRI/Notl adaptor primers and subcloned into the pAMP10 vector (Life Technologies, Gaithersburg, Md.) to yield libraries of selected cDNAs.

Recombinant clones were arrayed on blots. Clones that hybridized to either repetitive or ribosomal sequences were excluded from further analysis. To confirm their origin, unique clones were individually hybridized to EcoRI digests of cosmid/BAC/P1 DNAs and DNAs from chromosome 16-specific human-hamster hybrid lines. Clones were then hybridized to each other and were binned into groups. Representative clones of each group were hybridized to multiple tissue Northern blots and sequenced.

cDNA Identification by Solution Hybridization

Following the protocol provided in the Gene Trapper kit, the furthest centromeric cDNA, clone v75-1, was isolated by solution hybridization of a leukocyte cDNA library with biotinylated oligonucleotide probes derived from 2 exons trapped from PAC 273L24. Solution hybridization was carried out using the GeneTrapper cDNA Positive Selection System (Life Technologies, Gaithersburg, Md.).

Two trapped exons, v66 and v75, were used as starting material. PCR screening of Superscript cDNA libraries (Life Technologies, Gaithersburg, Md.) derived from human brain, liver, leukocytes, spleen, and testis were used to determine the tissue-specific expression of these exons. GeneTrapper experiments were performed with sense and antisense primers from both exons, assuming both orientations of these exons in the putative transcript.

The following oligonucleotides were synthesized and PAGE-purified:

v66GTI: AAG CTC ACT GCC TTC TCC TC; (SEQ ID NO:28)

v66GT2: GAG GAG AAG GCA GTG AGC TT; (SEQ ID NO:29)

v75GTI: GAC TTG GAA ACA AGT GGG AG; (SEQ ID NO:30)

v75GT2: CTC CCA CTT GTT TCC AAG TC. (SEQ m NO:31).

Oligos were biotinylated, hybridized to single-stranded DNA from the leukocyte cDNA library (one primer per reaction), followed by cDNA capture using paramagnetic streptavidin beads and repair using the corresponding non-biotinylated oligos. Colony hybridization of lifts using ³²P-dCTP end-labeled oligos was used to identify positive clones. Gel-purified inserts from these clones were hybridized to cosmid contig blots in order to distinguish cDNA clones mapping to the FMF region from false positive clones due to homologous domains. All positive clones were identified by the primers v66GT2 and v75GT2, and no clones were identified by the other set of primers.

Characterization of cDNA v75-1

The translated v75-1 cDNA sequence is shown in FIG. 2. The exon-intron structure deduced from the genomic sequence of two cosmids is depicted in FIG. 3C. Shaded boxes represent exons; introns are drawn to scale. The numbers above the boxes represent the size of the exons in bp. The numbers below the boxes reflect the order of the exons with 1 being the most 5′.

Although there is an excellent Kozak consensus (Kozak, supra) at the initial methionine, the reading frame remains open in the cDNA upstream. There are no splice-acceptor consensus sequences or in-frame methionines with good Kozak sequences before the first stop upstream in the genomic DNA. Additionally, the transcript size by Northern blot is 3.7 kb. The estimated transcript size from cDNA is 3503 nucleotides. Therefore, the sequence appears to be the full-length sequence.

Example 2 Mutational Analysis

Three different v75-1 mutants of FMF carrier chromosomes in multiple ethnic groups are not seen in a panel of almost 300 normal control chromosomes. This indicates that v75-1 is a cDNA of MEFV, the gene associated with FMF.

Three missense mutations were identified in exon 10 of v75-1 (FIG. 5) after screening a total of 165 individuals from 65 families. All three mutations are clustered within 46 amino acids of one another in the highly conserved rfp (B30.2) globular domain at the C-terminal end of the predicted protein. The first mutation, is a G C transversion at nucleotide 2040 in which methionine is replaced by isoleucine (M680I). This mutation was observed in the homozygous state in the affected offspring of a single Armenian family. The second mutation is a A G transition at nucelotide 2080 in which methionine is replaced by valine (M694V). This was observed in a large number of affected individuals bearing four apparently distinct disease associated haplotypes. The third mutation is a T C transition at nucleotide 217 which substitutes alanine for valine (V726A). It was observed in affected individuals bearing the C haplotype in a Druze family and in other FMF patients and carriers bearing this haplotype. An additional mutation in which lysine is replaced by arginine at positions 695 (K695R) was observed in an American FMF patient of Northern European ancestry.

Direct sequencing of RT-PCR products or amplified exons from the 8 cDNAs telomeric to v75-1 failed to identify disease-associated mutations.

It is extremely unlikely that the substitutions in v75-1 are actually polymorphisms in tight linkage disequilibrium with “real” mutations on a nearby gene. This hypothesis would require that there be 3 such v75-1 polymorphisms on 3 different haplotypes, each in perfect linkage disequilibrium with the mutations on the “real” FMF gene. While not impossible, such a scenario is at least unnecessarily complex. It is also unclear where such a closely linked gene would be located. The historical recombinants at the 5′ (centromeric) end of v75-1 exclude the interval between D16S33 73 and v75-1. On the telomeric side, the 5′ end of a novel zinc finger gene is located within 10 kb of the 3′ end of v75-1, but thorough screening has revealed no mutations in this later gene (data not shown). Moreover, there are no trapped exons, direct selected cDNAs or expressed sequence tag (EST) hits that map to the interval between them. Finally, and most importantly, the observation of normal chromosomes that bear disease-associated microsatellite and SNP haplotypes but do not have the M680I, M694V or V726A mutations is strong evidence that these are not just haplotype-specific polymorphisms.

Mutation Detection by Fluorescent Sequencing

The entire coding region was sequenced, plus splice cites, in individuals representing seven microsatellite haplotypes. Approximately 100 ng of genomic DNA template was used in PCR reactions to amplify exons and flanking intronic sequences according to the supplier's recommendations for AmpliTaq Gold (Perkin Elmer, Branchburg, N.J.) and Advantage-GC Genomic PCR Kit (Clontech, Palo Alto, Calif.).

The PCR primers were tailed with one of the following sequences:

21 M13 forward: GTA AAA CGA CGG CCA GT; (SEQ ID NO:32)

28 M13 reverse: CAG GAA ACA GCT ATG ACC AT; (SEQ ID NO:33)

40 M13 forward: GTT TTC CCA GTC ACG ACG. (SEQ ID NO:34).

After amplification, reactions were run on 1% agarose gels and gel purified using either QIAquick gel extraction kit (QIAGEN, Santa Clarita, Calif.) or Microcon/Micropure/Gel Nebulizer system (Amicon, Beverly, Mass.). Alternatively, PCR products were column purified with Microcon-100 (Amicon). Purified amplicons were sequenced with dye primer chemistry (PE Applied Biosystems, or Amersham, Cleveland, Ohio). Sequencing reactions were ethanol precipitated and run on an ABI 377 automatated sequencer. Sequence data were analyzed with either Autoassembler 1.4 (PE Applied Biosystems, Branchburg, N.J.) or Sequencher 3.0 (Gene Codes Inc., Ann Arbor, Mich.).

Example 3 Protein Modeling

The deduced amino acid sequence was examined. Two overlapping nuclear targeting signals were detected using the PSORT algorithm (Nakai and Kanehisa, “A knowledge base for predicting protein localization sites in eukaryotic cells,” Genomics, 14:897-911 (1992). The first nuclear targeting signal is a four residue pattern composed of a histidine and three lysines. The second is a Robbins/Dingwall consensus (Robbins et al., “Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence,” Cell, 615-523 (1991). A bZIP transcription factor basic domain (Shuman et al., “Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP, Science, 249:771-774 (1990) was identified using a PROSITE search (Bairoch et al., “The PROSITE database, its status in 1997,” Nucleic Acid Res., 25:217-221 (1997)). The spacing of cystine and histidine residues between residues 375 and 407 (denoted by plus signs in FIG. 2) resembles a B-box type zinc finger domain (Reddy et al., “A novel zinc finger coiled-coil domain in a family of nuclear proteins,” Trends Biochem. Sci., 17:344-345 (1992)).

Example 4 Localizing Expression of the Protein

The tissues in which v75-1 is expressed are highly consistent with the clinical phenotype for FMF. Based on the nature of the inflammatory infiltrate and the anatomic localization of inflammation in FMF, MEFV gene expression might be predicted to be observed in granulocytes and/or serosal cells. Multiple tissue northern blots demonstrated high levels of expression in peripheral blood leukocytes, primarily in mature granulocytes, but not in lymph nodes, spleen or thymus which are comprised largely of lymphocytes.

FIG. 4 shows the expression profile for the v75-1 gene. FIG. 4A shows the results of hybridization of a probe derived from exon 2 on multiple tissue Northern blots. A 3.7 kb transcript was found in peripheral blood leukocytes (PBL) and colorectal adenocarcinoma (SW480). The presence of the transcript in peripheral blood leukocytes compare favorably with the symptoms associated with FMF. The detection of the 3.7 transcript in colorectal adenocarcinoma is unexplained.

FIG. 4B shows hybridization of the same exon 2 probe on Northern blots with mRNA from purified Polymorphonuclear leukocytes (PMNs) and lymphocytes. PMN lanes represent preparations from different individuals. A β-actin control can be seen at the base of the gel.

The following abbreviations were used in FIG. 4: HL-60 (promyelocytic leukemia); K-562 (erythroleukemia); MOLT4 (lymphoblastic leukemia); A549 (lung carcinoma); and G361 (melanoma).

Northern Blot Analysis

To determine transcript size and level of expression in various tissues, multiple tissue Northern blots (Clontech) were hybridized with probes derived from various exons of the gene. These exons were amplified and purified as part of the sequencing protocol for mutation analysis. Larger exons (2, 5, and 10) were labeled by random-priming using Stratagene Prime-It Kit and ³²P-dCTP (ICN). Hybridization and washing of blots were essentially as described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (1989), except using Hybridisol I (Oncor) prepared hybridization buffer. Hybridization was detected by autoradiography, with 4 hour exposures. Northern blots with mRNA from highly purified peripheral blood lymphocytes, PMNs, and monocytes were the kind gift of Drs. H. Lee Tiffany and Harry Malech.

Example 5 Homologies to Other Proteins

FIG. 6 shows the alignment of the rfp (B30.2) domain of pyrin with homologous proteins. The following abbreviations are used in FIG. 6: hum-RFP (RET finger protein; SWISS-PROT P14373); xla-xnf7 (nuclear phosphoprotein xnf7, Xenopus laevis; PIR A43906); pwa-A33 (zinc-binding protein A33, Pleurodeles walt1; SWISS-PROT Q02084); hum-SS-A/Ro (52 kDa RO protein;

SWISS-PROT P19474); hum-afp (acid finger protein; GenBank U09825); hum-BT (butyrophilin; GenBank U90552); hum-efp (estrogen-responsive finger protein; PIR A49656); hum-B30-2 (B30-2 gene; PRF 2002339); pig-RFB30 (ring finger protein RFB30, Sus scrofa; EMBL Z97403); hum-Staf-50 (transcription regulator Staf-50; IR A57041).

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. All publications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

45 1 16891 DNA homo sapiens misc_feature (0)...(0) n = a, t, c, or g 1 tatttttgta ttttagtaga gatggggttt actgtgttgg ccaggctggt cttgtactcc 60 caacctgagg tgatccaccc acctcggcct cccaaagtgc tgggattaca ggcgttagca 120 ctgtgccctg cccccaacat gtaacttctg ttagcttcaa agccacctct ggggccctgc 180 accacatatg agctgaagga cacccgtgcc ttttcacccg tgtagctcca gcatcttggc 240 acactgtcta gaatgttcaa tgaatgtgca cggaagagca ttctggctcc agggagcgag 300 gactgagtca gctctgggaa cagatgagtc aggctggtgg tccaggcatt gcttttcaag 360 tccttcatgt ggctggaaga accagtcaac tggaaccgga tcaacagggg tgatggcatg 420 gcaagagtta tctcctggca gtgcccttct ggcctcactt gccttcttgg gccaggaaag 480 gcaaagctca caggactgta ttcagtgccc accccttccc ccgtcctgtg ccattggctc 540 tggaaggtcc ctgaaacccc gagtctggag gagaacagtt gaccagcagg gcgggccctc 600 agcatagtcc tctctgttcc cactcacccg ctctgccagc cccagatcct ggcaggaagg 660 aagattggag ggggtgtctg gaatccaatc ccagaccttc ccttgcagac ttgcccatct 720 gtctgtggtc tagtgtggag gcgaggtcca gggtttggga ggggtgtggg ggcacatgtc 780 tgccaaggca tggagccctc ccagctggaa aatcctctga acctgtaaga agagaacaca 840 gccggcatgg acacaccctt acccttagtc tcagttccca ccaagacaca gagcatttcc 900 tgtgcctttt ccgctatttc acaacctgcc ttttcttgct caccaaggac agaggcttct 960 tttcctacca gaagccagac agctggctcg agcctctcct gctcagcacc atggctaaga 1020 cccctagtga ccatctgctg tccaccctgg aggagctggt gccctatgac ttcgagaagt 1080 tcaagttcaa gctgcagaac accagtgtgc agaaggagca ctccaggatc ccccggagcc 1140 agatccagag agccaggccg gtgaagatgg ccactctgct ggtcacctac tatggggaag 1200 agtacgccgt gcagctcacc ctgcaggtcc tgcgggccat caaccagcgc ctgctggccg 1260 aggagctcca cagggcagcc attcagggta agcgggccca ggcctcctcc tcatccagtg 1320 ctgagtgctg gctgctttgt gggaaagggg accaggagct cagagcagct cactctgacc 1380 tggggattgg gagtctcagg tctaccaaaa tccagatgac tttagttcag gaacgtccct 1440 ttcttcactc tggcctttgg aactgggtta gtaaacttcc ttcaggctcc taatgggttt 1500 tttaagaagc aggtcagggt cacgaaaggc aggagctgga acacctgttc tttgagactt 1560 cttcactaca tttatgatta atactcatgt cagacaaaca tctctaggtt agcaaaaagg 1620 gattgctatg caatcatatg aacggggttg gtatagaatc ttctcagtgc tgttcaccat 1680 gttggccagg ctggtctcga actcctgacc tcaagtgatc ctcccgcctc agcctcccaa 1740 agtgctggga tttcagacat aggccaccgt gcccggctta tttttatttt taaagcgtat 1800 aatctgggtt ttgctgacct gtgtaagatc ttatttgaaa cagttgtcct gcttaaaacg 1860 tttgaaaagt actatttgag aaatataggc taggcatggt ggctcacact tataaataat 1920 ctcagcactt tgggaggcta aggtgggtgg attgctagag ctcaggagtt tgagaccagc 1980 ttgggcaaca tggtgaaacc ctgtctctac caaaaataca aaaaaatgag ccaggcgtgg 2040 tagcacacac ctgtattttc agctattgaa aaaacagaaa acaggctgag gtgagaggat 2100 tgcttgagcc tgggaggcag aggttgcagt gagctgagat cacatcaggg caacagagca 2160 agatcctgtc tcaaaaaata aaataagaga gagagaaata catagcaaca tcaagcatgt 2220 tcttactgaa tggtaattga ctgccattgt ctagtctggg nagtcctgaa cttttgtttt 2280 tgagatggag tcttgctctg tcactcaggc tggagtgcag tggcccgatc tcagctcnct 2340 gcaacctcca catcccgggc tcaagcgatt ctcatgcctc agcctcccga gtagctggga 2400 ctacaggtgc gcaccaccgc gtctggctga gtttcttatt tttagtagga acggggtttt 2460 gccatgttgg ccaggctggt ctcgaactcc tgacctcaaa tgatcctccc accttggcct 2520 ctggagaagc tgggattaca ggcatgcgca ccacgctcag cttatttttg tatttttagt 2580 agagacgggg tttcaccctg ttggtcttga actcctgatc tcaggtgatc ctcccgcctc 2640 ggcctcccag agtgccggga atacaggcat gagccaccgc gcccggcccg ttgttttcct 2700 caatttctaa actttaatat ccaaggggat tctctctcct ctgccctgaa tcttgggccc 2760 taaacgtggg acagcttcat cattttgcat ctggttgtcc ttccagaata ttccacacaa 2820 gaaaacggca cagatgattc cgcagcgtcc agctccctgg gggagaacaa gcccaggagc 2880 ctgaagactc cagaccaccc cgaggggaac gaggggaacg gccctcggcc gtacgggggc 2940 ggagctgcca gcctgcggtg cagccagccc gaggccggga gggggctgtc gaggaagccc 3000 ctgagcaaac gcagagagaa ggcctcggag ggcctggacg cgcagggcaa gcctcggacc 3060 cggagcccgg ccctgccggg cgggagaagc cccggcccct gcagggcgct agaggggggc 3120 caggccgagg tccggctgcg cagaaacgcc agctccgcgg ggaggctgca ggggctggcg 3180 gggggcgccc cggggcagaa ggagtgcagg cccttcgaag tgtacctgcc ctcgggaaag 3240 atgcgaccta gaagccttga ggtcaccatt tctacagggg agaaggcgcc cgcaaatcca 3300 gaaattctcc tgactctaga ggaaaagaca gctgcgaatc tggactcggc aacagaaccc 3360 cgggcaaggc ccactccgga tggaggggca tctgcggacc tgaaggaagg ccctggaaat 3420 ccagaacatt cggtcaccgg taaattgtgt tctttccaac tttatatcgg ctgcagagaa 3480 agaatggctg gccgggcacg atagctcatg cctgtaatcc cagcgctttg ggaggccagg 3540 gcgggaggat tgctggaggc caagactttg agaccagcct ggtgaatgta gtgagacccc 3600 cgccatctct ataaacgaaa ttaaaaaaat aaaaacccaa aggttgggca gggcgtggta 3660 gctctcgcct gtaatcccag agctttgaga ggcctgcacg ggaggatctc ttgaccccag 3720 gagttccata ctagcctagg caacacagtg agaccccatc tctacaaaat acaatagtgg 3780 cacgcgcctg tagtcccagc tgctcgggtt cacttgagca gacggagttc caggctacag 3840 tgagctgagg atcatgccac tgcacaccag cctgagcaac gtagccagac tcacttctac 3900 aaaactaaaa aaaaaattag ctgggtatgg tggcacacgc ctgtaattct agccactcag 3960 gaagctgagg caggaggatt gcttgagcca gggagttcca ggctgcagtg agctgaggat 4020 gtgccactgc actccggcct gggcaacaga gcaagaccct gtctcttaaa cattttgggg 4080 ggaaaaaaaa agaaagaaag aatgtccgat tgaaaaaggc aatcaggtgt tatcagtggc 4140 caaagaatgg agaaggggag ctcacctctg caggcgtctg cttgccaggg atgggaggca 4200 gggcgatttt agagtccagg gaggggaagg gagataggta agcaggccca gggcagggtt 4260 ccatatgtgc aggcgctgtc cccagcatgc ttcttcctac atcgcattca aacaaaccct 4320 tctccatctt ctttagggga ggacccttta gcttataacc atgtgtaaat gatcctaagg 4380 taactggaag tcacctcttc cagtttgcac tggttttgct ctgatcttaa cttcctctgg 4440 tttttggcaa gggatcagga ggctccaggc catctggatt tttttaagca gctgtcccta 4500 taggtaaaga gactaaaaaa aaactgtaaa agaaaaatgc caccagttta gagggtaccg 4560 aggctatcca ggtgacaatt ccatgctcgt ggtgggggca gcattcagaa acacactttc 4620 cttttttttc ctcctttttt tttttgagac agagtctcag tctgtctccc atgctggagt 4680 gcagtagtgt gagcacagtt tactgcagcc tcaacctcct aggctcaagc gatcctccca 4740 cctcagcctt ccaagtagct gagactatag gtgctcacca ccacacctgg ttaatttttt 4800 tttttttttt tgtatttttt gtagttacga ggactgtcta tgttgcccag gctggttttg 4860 aactcttggg ctcaagcgat cccccgcctt agcctctaaa agtgctagga tttcaggtgt 4920 gagtcactac acccagccta tggaacacac tttccaatgc attgttggct ggagaggaga 4980 aatcacagca ctcaaggagg agaaatagaa ttgggggtcc aggccgggtg cggtggctca 5040 tacctgtaat cccagcactt tgggaggcca atgggggcgg atcacctgag gtgaggagtt 5100 cgagaccagc ctgccaacat ggtgaaacgc catctctact aaaaatacta aatttgctgg 5160 gcgtggtggc gggtgtccat aatcccagct actcagaagg cttcgaggca ggagaattgc 5220 ttgaaccgag gaggcagagg ttgcagtgag ccaagatcat gccactgcac tctagcctgg 5280 gcgacaagag caaaactctg tctcaaaaaa aaaaaaaaaa aagaattggg agtccaggga 5340 cccctgagac ctgggagggg aaaggatgtg gtatgctgca tgagtcttca aatccagaag 5400 tccctgggtc ttccagtgag aaaggaccct gggatctgga aaacctagca tccttaggaa 5460 tagtgacctg aaaagtactg aagtatttcc cccctaattt tcttttatcc ctactgtatt 5520 ttttttaatt tttttttttt tttagatatg gggtcttgct atgttgccca ggttggtctc 5580 gaactcctga tctcaaacaa tcctcccatc tttgcctccg aaactgctgg gattacaggt 5640 gtgcaccact gcaccaggtc cccactgtat ttatatcatt gggattcctg ggtgtcttct 5700 agggccgctt cgttaatctg atgcaggctt agaccctgaa aaatgcatat atgcacagct 5760 tcacaaatgt cacatcaaat ttcaggtagt tcttggacac tctgaagacc atctttagaa 5820 tccaaggggt ttatggacac caggtagaaa atctggggaa gactggttaa aaatactccc 5880 tctcacaata acctcacagc aatgcatcat catggggttg agattctacc attgcctttc 5940 tctcagcaga aagaaaagcc tattggctaa agtcctaact atctactgct gaggtagtca 6000 ttaaaattat gtttggttgt gaataataga aacacccaaa taacagtaac ctcaacagaa 6060 aagaagtttg tgcctccttc acataaatga tacacaggcg gtcccaggca gatccgtggg 6120 ccaggaccct ggggtcctgc tgttgctctg tcccaccaag tttgtcctca agcttctgct 6180 ctcagaaggt gacgtcctca tgccaggcag caagatggag gaacagaggg gaacagtatc 6240 cctcgggaaa gctctagaag tttctagaag ctgcttgtga cacctccatt tacatccctt 6300 tggtcatatt attgtcaaat agccacacct aactgcaaag gaggctgaga aatgcagggc 6360 atttgggggg caatgggagg cagggaaaca gggaaacgtg gacaattaat tctatcacga 6420 gagaaggagg gagagtaatt tctggtgact actagcagtc tcatttacag atgtgctgtg 6480 aatttctggg acactgtgag gtgggaggag gtagcagggg ctaaaggatt gagtgtgttt 6540 ctatttcttt ttttgttttt tttttttttg agatggagtc tctcttggtc acccagactg 6600 gagtgcagtg gcgcaacttc agctcactgc aaactccgcc tcccgggttc aagcaattct 6660 cctgcctcag cctcccgagt agctgggatt acaggtgccc accaccacgt ccggctaatt 6720 tttgtatttt tagtagagac agggtttcac catcttggcc aggctggtct tgaactcctg 6780 acctcatgac ccacccgcct cggcctccca aagtgctggg attacaggcg tgagccactg 6840 cgctcggcct tgtgtttcta tttcttcttg tatctcgtgg catgtctgct tatgaagttg 6900 caattagagt cttggagtag agctattcat aactgttagg tcttcatgat gagttccagt 6960 ctttagccct ataatgcccc ccttctttgc tttttctttt aagatggcat cttactctgt 7020 tgcccaggct ggagtgcagt ggtgcagcat caacctccta ggttcaagca atcctcctgt 7080 ctcagcctcc caagtagctg ggattagagg tgtgcaccac cacacctggc taatttttta 7140 attttttgta gaggtgggct cttgccatgt tgcccaggct ggtctcaaac tcctgagctt 7200 aagcagtcct cccaccttgg cctcccaaag cactgggatt ataggcatga gccaccaccc 7260 agccccttct ttgctttcat ttaatggtta ttgaactcat atgtgagcag tggtctattt 7320 attccttcat tcaatactca ttttccaaat gcttgcattt gccaggtact ctgctagggg 7380 ctgggatcca gctaggagcg aggtacacaa gtcaccatcc cctggaagcc tccactcacg 7440 ttatgggcag ccagggatgg gttcaagtgg caaaggaaca ctggtcagaa tgtctctttc 7500 cttggcatca cctgctagat ctatgtctgt gcaggaggaa cagcacaagg ccatgggtct 7560 ttctttagga taaatgccca agaattccaa ggctcaggaa tgtctgaggt ctggccctta 7620 gctctcaggc ccagtggcct gtttgcttcc tcactggatg gaagtcgggg gaggacaagc 7680 taggaagtgg gcagagtcta actgagaact cgcacatctc aggcaagggc tgtgtccgct 7740 gtgctttgtg atacctctgt gtaagcaact tgggtttgcc attcaggggg tttttccact 7800 gcatgtcccc aggaaggcca ccagacacgg ctgcgagtcc ccgctgccac gcccaggaag 7860 gagacccagt tgacggtacc tgtgtgcgtg attcctgcag cttccccgag gcagtttctg 7920 ggcaccccca ggcctcaggc agccgctcac ctggctgccc ccggtgccag gactcccatg 7980 aaaggaagag cccgggaagc ctaagccccc agcccctgcc acagtgtaag cgccacctga 8040 agcaggtcca gctgctcttc tgtgaggatc acgatgagcc catctgcctc atctgcagtc 8100 tgagtcagga gcaccaaggc caccgggtgc gccccattga ggaggtcgcc ctggaacaca 8160 aggtaggcac tccctgcctg tgggctcttc tctgccaggc acttggacac actgggcctt 8220 acttcatttt cccaacaact ctgggttgtt ggtgcattaa ccagcattct tgggctggaa 8280 atggcaagaa cacaatataa accagtccag caaagagggg agctacaggt ttatgttgct 8340 cagagatcca gggggagctg gcttcaggta tggctgaatc cagaggctca gaggaagtgc 8400 ctctcagctc tgctgccttt ggcaattcag ccattcctcc ctcctctttc ctgagcaccc 8460 ctccccatgc cgctggcagc agcaccctca gccttgctac cagaaggaga tgttcccctc 8520 cagagttggc accagctaaa gatggcagga gccaaattca agcttttcaa caagtgctgt 8580 ttttccagaa gaaaattcag aagcagctgg agcatctgaa gaagctgaga aaatcagggg 8640 aggagcagcg atcctatggg gaggagaagg cagtgagctt tctggtaagg tcagaggtgg 8700 ctgatggccc atccgtccct gggaggaagg tgggaagagt gagcaggggt ccccgagatt 8760 ctgctgtggt tcacagggca gcagggatgg ccacctcctc tcaggggaca gagggtaacc 8820 agcagccaag ggtaagctca tccctgtaga gggagaccac ccccagcagg caggggtcac 8880 ctctgaggat cctgtcatgc tttctcatac tcaccagaag atggtagaga gcaacctatg 8940 ccggtgacta ctgcagaaag atgggattga ggaaaaggga ggagaacgcc actttctttt 9000 tttgtgacgg agtctcgctc tgtcacccag gttgtagtgc agtggtgtga tcttggctca 9060 ctgcaacctc tgcctcccgg gttcaagcga ttctcctgcc tcagcctcct gagtagctgg 9120 gattataggt gagtgccacc atgcctggct aatttttgta gttttagtag agatggggtt 9180 tcaccatgtt ggtcaggctg ttctcgaact cctgaactcg tgatccgccc gccttggcct 9240 cccaaagtac tgggattaca gatgtgagcc actgcgcccg gccaagaaca cttttaactt 9300 cataatttac tctctgtttt tttgttttgt ttccaagatg gagtctcgct ctgtcaccca 9360 ggctggagta cagtggcacg atcttggctt gctccaacct ccacctccga ggttcaagca 9420 attctcctgc ctcagcctcc ttagtggctg gaattacagg cgcctgccac cgcgcctggc 9480 taatttttgt atttttagta gagacgggat ttcaccgtgt tggccaggct ggtctcaaac 9540 tcctgacctc aggtgatcca cctgcctcgg cctcccaaag tgctgggatt acaggtgtga 9600 gccatcgtgc ctgggctggt ttttttgttt tttagggttt tttttttttt ttttttttga 9660 gatggaatct cactccgtcg tccaggctgg ggtgcagtgg tgcaatctcg gctcactgca 9720 aaccttcgcc tccccagttg aagcaattct cctgcctcag cctcccgagt tgctgggact 9780 gtaggcacat gccaccactc ctggctaatt tttgtatttt tagtaaagac agagtttccc 9840 catgttggcc aggctggtct cgaactcctg atctcaagtg atctgcccaa ctcagcctcc 9900 caaagtgctg ggattacaga catgagccaa tgcacccagc ccaaatttcc ccattttata 9960 agacaacatt tatattggat tagggaccca cccaatccca gtaggaccac atcttaacta 10020 attacatctg caagaactct tatctccaaa taagatcaca tgctgagtac tgggggttag 10080 ggcttcaacg tgtaaatttt ggaagggaca cagttaaacc ttaacaccag gtttaaggac 10140 attttcccag agctagcccc agccatgctc agtcttttct ggaaggttcc agacaatatc 10200 gcctcctgct ctggaatcta ggccttgaag aggcagcata agcccacctc ttatccacct 10260 ccaggaggtg ggcttctggg ggttcctgga catccacgtc cacccacagc acagaccccc 10320 atacctccct gtcctctgct ccccagaaac aaactgaagc gctgaagcag cgggtgcaga 10380 ggaagctgga gcaggtgtac tacttcctgg agcagcaaga gcatttcttt gtggcctcac 10440 tggaggacgt gggccagatg gttgggcaga tcaggaaggc atatgacacc cgcgtatccc 10500 aggacatcgc cctgctcgat gcgctgattg gggaactgga ggccaaggag tgccagtcag 10560 aatgggaact tctgcaggtg ggtgtgcctg ggcccggctt tcttgggtcc cctgtgccta 10620 tcaggatgcc tcaggctccc agctctgcca tcagccgtgc tggaacaagt gggtgaagcc 10680 ctaaggccta ggataggact tggtcttggt gacccacagt gcctcttgtg cccagacccc 10740 tttgatgagg tctctcagga gcccagggtg gcctggtatc caggggatct ctgccatttc 10800 ccagaaggga tcagcagggc ttgagggccg ttccattgca ggcctcgcca cctgggatgc 10860 ctgaattccc gtggttagaa ttagacttga agaaaggtgc tccacttcca ctgacaccct 10920 agggcaggga gccctggtaa gtgcagcggg gagctaaaag tccaggagcc cagaagtaga 10980 ggccaggagt cagcccagcc actaggagcc tggtaaccga cagtttcctt cttttttctc 11040 ctaggacatt ggagacatct tgcacaggta cagcgaggtc ctgtggtgta ccctggggtg 11100 tcttgcagaa agcatatggg ggagacagtc ccagaaggga cctgggaggg agatgttccc 11160 aaccccgggg tctgtgattc cagactcctc cttttttctg cagcttccca aagcctctct 11220 ggatttgata gggagaaggg catctggtca gcagggaggc tggccgggta tggagctgca 11280 gactgggaag ggtgaattca gcccatcctg ctgaaacaag atggaggctc cctaagaaac 11340 cttccgagtg cattgtgtcc cgtgcagttc atctgatgaa agctgcccct tcaggcctac 11400 tggtggcctt gggaagcttg tttggagtgg agctgggcta agcccagcag gaaggggagg 11460 ggagggaagg gacaggaaga ggctaagcct taaaatcacc tgggagcttt acaaaatccc 11520 ggtgtccttt tgtgtctggc ttcttcactt agcataatgt cttcgggctt catccgtgtt 11580 gtaacgtgta tcagaattta ttttcttttt atggctgaat catagtccag tgtgtgttca 11640 tacattttgc ttatccattc atggatatcg ggacttcttc taacttttgg tttgtgaata 11700 atgttgctat gaacaagggt gtacaaatat ctgcttgaga ccctgctttg ttattttggg 11760 tacctaccca gaagtggaac tgcgggacca tgtggttatc ctgtgtttaa ttttttttga 11820 ggaaccacca tcctaattct cacaggggct gcatcgcttc acattcccac cagcagcaca 11880 caggggctcc agtttctcca catctttgcc atcacttatt ttcttctgtt tcactctctc 11940 tctctctctt tttttttgaa gacagcgtct tgctctgtca tccaggctgg agtgcagtgg 12000 cgcgatcttg gctcactaca acctctgcct cccaggttca agggattctc ccacctcagc 12060 ctccctagta gctgggacta caggagcgtg ccaccatgcc cagctaattt ttttggtaga 12120 cagggtttca ccatattagc caggctggtc tcaaactcct gacctcaagt gatccaccca 12180 ccttggcctc ccaaagcgct gggattgcag gcgtgagcac cgtgcccagc catttctctt 12240 tccttccttc cctccctccc tcccttcctt cctttcttcc ttccttcctt tcttttcttc 12300 ttgagacaag gtctcactcc catcactaag gctggagagc agtggcacag tcacagctca 12360 ctgcaggctc agcttcctgg gctcgggtga ttctgagtag ctggcatcct gagtagctgg 12420 gactacaggc atgtgctacc acttccggct acttttttgt atttttaata gagacagggt 12480 ttcgccatgt tgcccaagct ggacttgaac tcctgggctc aagcgatccc actgccccgg 12540 cctcctgaag tgctaggatt acaggcatga gccaccatac ctggtctatt tttttctgtt 12600 gttgctgttt ttataatagc cattctaatg gatgtgaagg gatattttgt tgtgtgtgtt 12660 tttttttcat ttattatctt tttatttcaa tagaaagaaa ggggtgtata atcaatttga 12720 catagataat tctagtagat aatatcaatg tcattttaag tccattctga aaactccttg 12780 tggttttgat atccatgtct ttaaagcacc ccagtacatg acagtctgtg gccaaagttg 12840 aggaccagca tttagacctc tgaatccagg gaagactttt ctttgtgtag ctcaggctgg 12900 gctaggtgtg ccttgtggag aatgtagttc atttccagct cacgggtact tgggccaccc 12960 cctcgctccg gccttctctg gtcaacagtc ttttgtctct agggctaaga cagtgcctgt 13020 scctgcaaag tggaccactc ctcaagagat aaaacaaaag atccaactcc tccaccagaa 13080 gtcagagttt gtggagaaga gcacaaagta cttctcaggt agatgggctt gggagaagat 13140 tggaggtgca tgctcacttc ctccctaaga tccacatagc ccagagcccc tcacttccct 13200 cctcttcccc tggtcttgct gacctgcctt caacctctcc tccatctgtc cctggctgag 13260 ggacctaact ccagcttctc tctgctccct ttcccacatt ttagaaaccc tgcgttcaga 13320 aatggaaatg ttcaatggtg agtccagcgg taatggtgtg tgctggcctg gggttgttgc 13380 agtgttccct tgtgctgttg acttgagggg ccctatttag aagacaaaaa aaaaaaccaa 13440 acacctggag caaaggtagg agaaaggtca tggcaggccc cccaggctct gtgcgtgact 13500 cattgactga gttgactcat tagaccacag tccccaacat ggcctgggtt cctgggagga 13560 acgggattat acccaacata gcatgcaggg ccctaagcag ggggttcctt gtctttcctt 13620 gttgtcagga cagtgtaatt tagcccctct taatgctaat gctcaggaat tttttcccta 13680 tctgattttt ctccgtagtt ccagagctga ttggcgctca ggcacatgct ggtaagtgcc 13740 cagatcaagg caagtggccc tggcctgctg gatccctgtg ctctccccta ccacgttcca 13800 gaagaactac cctgtccctg tttcctgcag gtggggagaa ccctgtaggg atgttgccca 13860 tggaccccta cctaggtatt caaattttct ttgcagttaa tgtgattctg gatgcagaaa 13920 ccgcttaccc caacctcatc ttctctgatg atctgaagag tgttagactt ggaaacaagt 13980 gggagaggct gcctgatggc ccgcaaagat ttgacagctg tatcattgtt ctgggctctc 14040 cgagtttcct ctctggccgc cgttactggg aggtggaggt tggagacaag acagcatgga 14100 tcctgggagc ctgcaagaca tccataagca ggaaagggaa catgactctg tcgccagaga 14160 atggctactg ggtggtgata atgatgaagg aaaatgagta ccaggcgtcc agcgttcccc 14220 cgacccgcct gctaataaag gagcctccca agcgtgtggg catcttcgtg gactacagag 14280 ttggaagcat ctccttttac aatgtgacag ccrgatccca catctataca ttcgccagct 14340 gctctttctc tgggcccctt caacctatct tcagccctgg gacacgtgat ggagggaaga 14400 acacagctcc tctgactatc tgtccagtgg gtggtcaggg gcctgactga atgcccaaca 14460 ctgcatctct cttcctgctt ctggccttgt atcttgcatt cacactcaat agtcacggaa 14520 tgccgactag gtgctagctg ctatgggaaa tgcmaaaata acaaaatagt tactgtgccc 14580 acggagccct acccgattat agcagaggta agttaggaac gaacatgtta gtcaatccgg 14640 gtgaagacat gtactgatga cacaccatgg atttcagagg aggaagtacg gagtcgttgc 14700 ataatccgcc cctggtgggt ggcactctca ggtgctcctg aacagaagat ttggccctca 14760 ttttccctca gaaccccacg gcaaggatat atgtcccctt gttctctctg cttctgtctt 14820 gaggatatgg gaagcctaga gaaacgcaag cagactggat tgggatagaa gtatttgtgt 14880 acctggatta atgaactatg attttttttt tttttttttg agaccaaatc ttgctctgtg 14940 gcccaggctg gagtgcagtg gcacgatctc agctcactgc aacctccacc tcccaggttc 15000 aagcgattct cctgcctcag cctcctgagc agctggggat tacaggtgcg tgccaccaca 15060 ccaggctggt tttcttgtat ttttagtaga gacgggggtt tcaccatgtt agccaggctg 15120 gtctcgaact cctgacctca ggtgatccac ccgcctcagc ctcccaaagt gctgggatta 15180 caggcatgag ccactgtgcc cggcctatga ttcttttttt tttttttttt tgagacaaag 15240 ttttgctctt gtcacccagg ctggagtgca gtggtgcaat cttggctcgc aacctccgcc 15300 tcccaggttc aagagattct cctgcctcag cctccgaagt agctgggatt acaggcgccc 15360 gccaccatgc ccggctaatt ttttgcattt ttagtagaca tgaggtttca tcatgttggc 15420 caggccggtc tcaaactcct gacctcaggt gatgcaccca cctcagcctc ccaaagtgca 15480 gggattacag gcatgagcca ccatgccggg ccatgattct taagagaatt gactgggcct 15540 catgaataaa aaaattagaa aatctggtca tttgcatttg tcactcaatc actgtggaat 15600 cccatttccc gactgcattt ncaggaagtc agatgggact actgtcatgg aaaaacattt 15660 gggcatgtta tttccaagtg tcagattatt ctgtcttggt ttgtatggga aaatctgcgg 15720 gttgtggaat attaggttct acttcacaca catcccgtgc atttgtcctt catttaaaga 15780 gatgtaaagg ggccgggcat ggtgactcac atctgtaatc tcagcatttt gggaggcaaa 15840 ggcgggtgga tcgcctgagc ccagggattg agaccagctg ggcaatgtgg cgaaaacccg 15900 tctctacaaa aaatacaaaa attagccata gggatggggg tgggaggatg gcttgagcgc 15960 aggagatcga ggctgcagca gtgaactgag actgcactac ggcaatccag cctgggcaac 16020 agagtgagtc cctgtctcca aaaagtggat gttaggagta caaaaatcaa atgaagatta 16080 gatccaaact cctatgccaa ctcctctgtc ttcactacta gagtgtagat tagactcaga 16140 tactccatgg ctatgatgag agcaggtaaa cttgctgggc tttcctccac gagttttatt 16200 ctataagagt aatccacatc ccaggacagt tcacatgacc tacggcttag ctgttccctg 16260 cggtgggtca tgtcttattc ccgattctcc cttgttataa gcttttcatg aatatctttg 16320 tgtatatttt ccaccacctc accatataca tatttttttc tcctgtgtta ttcctaaaat 16380 ggttcctgaa tgtgaaatat ctgataatgc ttcctacggg ttgccatacc atcctttgca 16440 aagattttta aaatatttca tgcccaaagc aatgactgcc atttaaaatt tttttgctga 16500 tttaataggg atgtaatgag gccttacttc tgttttattt cattacctgt taatgaggct 16560 gtgaattttt ccatgtgaat ttctgctttt tgcttcattc tatggaaatt gtacagttcc 16620 tttgaatact tgctatttgg aatctacata ttgaatttcg tgttttgctg tacttcctca 16680 ttacatggtt ttaggctggg tgcggtgctc acgcctgaaa tcccaacatt ttgggagccg 16740 gaggtgggca ggatcggttg gcaatcgagg gtttcgagac cgagcctggg cagacatggc 16800 gaaacctcgc cctctaccta gaaagataaa caaattagcg caggcaatgg tggtgagcac 16860 ctgtagtcct agctgataag gtctaggttg a 16891 2 3470 DNA homo sapiens 2 atggctaaga cccctagtga ccatctgctg tccaccctgg aggagctggt gccctatgac 60 ttcgagaagt tcaagttcaa gctgcagaac accagtgtgc agaaggagca ctccaggatc 120 ccccggagcc agatccagag agccaggccg gtgaagatgg ccactctgct ggtcacctac 180 tatggggaag agtacgccgt gcagctcacc ctgcaggtcc tgcgggccat caaccagcgc 240 ctgctggccg aggagctcca cagggcagcc attcaggaat attccacaca agaaaacggc 300 acagatgatt ccgcagcgtc cagctccctg ggggagaaca agcccaggag cctgaagact 360 ccagaccacc ccgaggggaa cgaggggaac ggccctcggc cgtacggggg cggagctgcc 420 agcctgcggt gcagccagcc cgaggccggg agggggctgt cgaggaagcc cctgagcaaa 480 cgcagagaga aggcctcgga gggcctggac gcgcagggca agcctcggac ccggagcccg 540 gccctgccgg gcgggagaag ccccggcccc tgcagggcgc tagagggggg ccaggccgag 600 gtccggctgc gcagaaacgc cagctccgcg gggaggctgc aggggctggc ggggggcgcc 660 ccggggcaga aggagtgcag gcccttcgaa gtgtacctgc cctcgggaaa gatgcgacct 720 agaagccttg aggtcaccat ttctacaggg gagaaggcgc ccgcaaatcc agaaattctc 780 ctgactctag aggaaaagac agctgcgaat ctggactcgg caacagaacc ccgggcaagg 840 cccactccgg atggaggggc atctgcggac ctgaaggaag gccctggaaa tccagaacat 900 tcggtcaccg gaaggccacc agacacggct gcgagtcccc gctgccacgc ccaggaagga 960 gacccagttg acggtacctg tgtgcgtgat tcctgcagct tccccgaggc agtttctggg 1020 cacccccagg cctcaggcag ccgctcacct ggctgccccc ggtgccagga ctcccatgaa 1080 aggaagagcc cgggaagcct aagcccccag cccctgccac agtgtaagcg ccacctgaag 1140 caggtccagc tgctcttctg tgaggatcac gatgagccca tctgcctcat ctgcagtctg 1200 agtcaggagc accaaggcca ccgggtgcgc cccattgagg aggtcgccct ggaacacaag 1260 aagaaaattc agaagcagct ggagcatctg aagaagctga gaaaatcagg ggaggagcag 1320 cgatcctatg gggaggagaa ggcagtgagc tttctgaaac aaactgaagc gctgaagcag 1380 cgggtgcaga ggaagctgga gcaggtgtac tacttcctgg aacagcagga gcatttcttt 1440 gtggcctcac tggaggacgt gggccagatg gttgggcaga tcaggaaggc atatgacacc 1500 cgcgtatccc aggacatcgc cctgctcgat gcgctgattg gggaactgga ggccaaggag 1560 tgccagtcag aatgggaact tctgcaggac attggagaca tcttgcacag ggctaagaca 1620 gtgcctgtcc ctgaaaagtg gaccactcct caagagataa aacaaaagat ccaactcctc 1680 caccagaagt cagagtttgt ggagaagagc acaaagtact tctcagaaac cctgcgttca 1740 gaaatggaaa tgttcaatgt tccagagctg attggcgctc aggcacatgc tgttaatgtg 1800 attctggatg cagaaaccgc ttaccccaac ctcatcttct ctgatgatct gaagagtgtt 1860 agacttggaa acaagtggga gaggctgcct gatggcccgc aaagatttga cagctgtatc 1920 attgttctgg gctctccgag tttcctctct ggccgccgtt actgggaggt ggaggttgga 1980 gacaagacag catggatcct gggagcctgc aagacatcca taagcaggaa agggaacatg 2040 actctgtcgc cagagaatgg ctactgggtg gtgataatga tgaaggaaaa tgagtaccag 2100 gcgtccagcg ttcccccgac ccgcctgcta ataaaggagc ctcccaagcg tgtgggcatc 2160 ttcgtggact acagagttgg aagcatctcc ttttacaatg tgacagccag atcccacatc 2220 tatacattcg ccagctgctc tttctctggg ccccttcaac ctatcttcag ccctgggaca 2280 cgtgatggag ggaagaacac agctcctctg actatctgtc cagtgggtgg tcaggggcct 2340 gactgaatgc ccaacactgc atctctcttc ctgcttctgg ccttgtatct tgcattcaca 2400 ctcaatagtc acggaatgcc gactaggtgc tagctgctat gggaaatgca aaaataacaa 2460 aatagttact gtgcccacgg agcctacccg attatagcag aggtaagtta ggaacgaaca 2520 tgttagtcaa tccgggtgaa gacatgtact gatgacacac catggatttc agaggaggaa 2580 gtacggagtc gttgcataat ccgcccctgg tgggtggcac tctcaggtgc tcctgaacag 2640 aagatttggc cctcattttc cctcagaacc ccacggcaag gatatatgtc cccttgttct 2700 ctctgcttct gtcttgagga tatgggaagc ctagagaaac gcaagcagac tggattggga 2760 tagaagtatt tgtgtacctg gattaatgaa ctatgatttt tttttttttt ttttgagacc 2820 aaatcttgct ctgtggccca ggctggagtg cagtggcacg atctcagctc actgcaacct 2880 ccacctccca ggttcaagcg attctcctgc ctcagcctcc tgagcagctg ggattacagg 2940 tgcgtgccac cacaccaggc tggttttctt gtatttttag tagagacggg ggtttcacca 3000 tgttagccag gctggtctcg aactcctgac ctcaggtgat ccacccgcct cagcctccca 3060 aagtgctggg attacaggca tgagccactg tgcccggcct atgattcttt tttttttttt 3120 tttttgagac aaagttttgc tcttgtcacc caggctggag tgcagtggtg caatcttggc 3180 tcactgcaac ctccgcctcc caggttcaag agattctcct gcctcagcct ccgaagtagc 3240 tgggattaca ggcgcccgcc accatgcccg gctaattttt tgcattttta gtagacatga 3300 ggtttcatca tgttggccag gccggtctca aactcctgac ctcaggtgat gcacccacct 3360 cagcctccca aagtgcaggg attacaggca tgagccacca tgcctggcca tgattcttaa 3420 gagaattgac tgggcctcat gaataaaaaa attagaaaat ctaaaaaaaa 3470 3 781 PRT homo sapiens 3 Met Ala Lys Thr Pro Ser Asp His Leu Leu Ser Thr Leu Glu Glu Leu 1 5 10 15 Val Pro Tyr Asp Phe Glu Lys Phe Lys Phe Lys Leu Gln Asn Thr Ser 20 25 30 Val Gln Lys Glu His Ser Arg Ile Pro Arg Ser Gln Ile Gln Arg Ala 35 40 45 Arg Pro Val Lys Met Ala Thr Leu Leu Val Thr Tyr Tyr Gly Glu Glu 50 55 60 Tyr Ala Val Gln Leu Thr Leu Gln Val Leu Arg Ala Ile Asn Gln Arg 65 70 75 80 Leu Leu Ala Glu Glu Leu His Arg Ala Ala Ile Gln Glu Tyr Ser Thr 85 90 95 Gln Glu Asn Gly Thr Asp Asp Ser Ala Ala Ser Ser Ser Leu Gly Glu 100 105 110 Asn Lys Pro Arg Ser Leu Lys Thr Pro Asp His Pro Glu Gly Asn Glu 115 120 125 Gly Asn Gly Pro Arg Pro Tyr Gly Gly Gly Ala Ala Ser Leu Arg Cys 130 135 140 Ser Gln Pro Glu Ala Gly Arg Gly Leu Ser Arg Lys Pro Leu Ser Lys 145 150 155 160 Arg Arg Glu Lys Ala Ser Glu Gly Leu Asp Ala Gln Gly Lys Pro Arg 165 170 175 Thr Arg Ser Pro Ala Leu Pro Gly Gly Arg Ser Pro Gly Pro Cys Arg 180 185 190 Ala Leu Glu Gly Gly Gln Ala Glu Val Arg Leu Arg Arg Asn Ala Ser 195 200 205 Ser Ala Gly Arg Leu Gln Gly Leu Ala Gly Gly Ala Pro Gly Gln Lys 210 215 220 Glu Cys Arg Pro Phe Glu Val Tyr Leu Pro Ser Gly Lys Met Arg Pro 225 230 235 240 Arg Ser Leu Glu Val Thr Ile Ser Thr Gly Glu Lys Ala Pro Ala Asn 245 250 255 Pro Glu Ile Leu Leu Thr Leu Glu Glu Lys Thr Ala Ala Asn Leu Asp 260 265 270 Ser Ala Thr Glu Pro Arg Ala Arg Pro Thr Pro Asp Gly Gly Ala Ser 275 280 285 Ala Asp Leu Lys Glu Gly Pro Gly Asn Pro Glu His Ser Val Thr Gly 290 295 300 Arg Pro Pro Asp Thr Ala Ala Ser Pro Arg Cys His Ala Gln Glu Gly 305 310 315 320 Asp Pro Val Asp Gly Thr Cys Val Arg Asp Ser Cys Ser Phe Pro Glu 325 330 335 Ala Val Ser Gly His Pro Gln Ala Ser Gly Ser Arg Ser Pro Gly Cys 340 345 350 Pro Arg Cys Gln Asp Ser His Glu Arg Lys Ser Pro Gly Ser Leu Ser 355 360 365 Pro Gln Pro Leu Pro Gln Cys Lys Arg His Leu Lys Gln Val Gln Leu 370 375 380 Leu Phe Cys Glu Asp His Asp Glu Pro Ile Cys Leu Ile Cys Ser Leu 385 390 395 400 Ser Gln Glu His Gln Gly His Arg Val Arg Pro Ile Glu Glu Val Ala 405 410 415 Leu Glu His Lys Lys Lys Ile Gln Lys Gln Leu Glu His Leu Lys Lys 420 425 430 Leu Arg Lys Ser Gly Glu Glu Gln Arg Ser Tyr Gly Glu Glu Lys Ala 435 440 445 Val Ser Phe Leu Lys Gln Thr Glu Ala Leu Lys Gln Arg Val Gln Arg 450 455 460 Lys Leu Glu Gln Val Tyr Tyr Phe Leu Glu Gln Gln Glu His Phe Phe 465 470 475 480 Val Ala Ser Leu Glu Asp Val Gly Gln Met Val Gly Gln Ile Arg Lys 485 490 495 Ala Tyr Asp Thr Arg Val Ser Gln Asp Ile Ala Leu Leu Asp Ala Leu 500 505 510 Ile Gly Glu Leu Glu Ala Lys Glu Cys Gln Ser Glu Trp Glu Leu Leu 515 520 525 Gln Asp Ile Gly Asp Ile Leu His Arg Ala Lys Thr Val Pro Val Pro 530 535 540 Glu Lys Trp Thr Thr Pro Gln Glu Ile Lys Gln Lys Ile Gln Leu Leu 545 550 555 560 His Gln Lys Ser Glu Phe Val Glu Lys Ser Thr Lys Tyr Phe Ser Glu 565 570 575 Thr Leu Arg Ser Glu Met Glu Met Phe Asn Val Pro Glu Leu Ile Gly 580 585 590 Ala Gln Ala His Ala Val Asn Val Ile Leu Asp Ala Glu Thr Ala Tyr 595 600 605 Pro Asn Leu Ile Phe Ser Asp Asp Leu Lys Ser Val Arg Leu Gly Asn 610 615 620 Lys Trp Glu Arg Leu Pro Asp Gly Pro Gln Arg Phe Asp Ser Cys Ile 625 630 635 640 Ile Val Leu Gly Ser Pro Ser Phe Leu Ser Gly Arg Arg Tyr Trp Glu 645 650 655 Val Glu Val Gly Asp Lys Thr Ala Trp Ile Leu Gly Ala Cys Lys Thr 660 665 670 Ser Ile Ser Arg Lys Gly Asn Met Thr Leu Ser Pro Glu Asn Gly Tyr 675 680 685 Trp Val Val Ile Met Met Lys Glu Asn Glu Tyr Gln Ala Ser Ser Val 690 695 700 Pro Pro Thr Arg Leu Leu Ile Lys Glu Pro Pro Lys Arg Val Gly Ile 705 710 715 720 Phe Val Asp Tyr Arg Val Gly Ser Ile Ser Phe Tyr Asn Val Thr Ala 725 730 735 Arg Ser His Ile Tyr Thr Phe Ala Ser Cys Ser Phe Ser Gly Pro Leu 740 745 750 Gln Pro Ile Phe Ser Pro Gly Thr Arg Asp Gly Gly Lys Asn Thr Ala 755 760 765 Pro Leu Thr Ile Cys Pro Val Gly Gly Gln Gly Pro Asp 770 775 780 4 543 DNA homo sapiens 4 accctgcgtt cagaaatgga aatgttcaat gttccagagc tgattggcgc tcaggcacat 60 gctgttaatg tgattctgga tgcagaaacc gcttacccca acctcatctt ctctgatgat 120 ctgaagagtg ttagacttgg aaacaagtgg gagaggctgc ctgatggccc gcaaagattt 180 gacagctgta tcattgttct gggctctccg agtttcctct ctggccgccg ttactgggag 240 gtggaggttg gagacaagac agcatggatc ctgggagcct gcaagacatc cataagcagg 300 aaagggaaca tgactctgtc gccagagaat ggctactggg tggtgataat gatgaaggaa 360 aatgagtacc aggcgtccag cgttcccccg acccgcctgc taataaagga gcctcccaag 420 cgtgtgggca tcttcgtgga ctacagagtt ggaagcatct ccttttacaa tgtgacagcc 480 agatcccaca tctatacatt cgccagctgc tctttctctg ggccccttca acctatcttc 540 agc 543 5 181 PRT homo sapiens 5 Thr Leu Arg Ser Glu Met Glu Met Phe Asn Val Pro Glu Leu Ile Gly 1 5 10 15 Ala Gln Ala His Ala Val Asn Val Ile Leu Asp Ala Glu Thr Ala Tyr 20 25 30 Pro Asn Leu Ile Phe Ser Asp Asp Leu Lys Ser Val Arg Leu Gly Asn 35 40 45 Lys Trp Glu Arg Leu Pro Asp Gly Pro Gln Arg Phe Asp Ser Cys Ile 50 55 60 Ile Val Leu Gly Ser Pro Ser Phe Leu Ser Gly Arg Arg Tyr Trp Glu 65 70 75 80 Val Glu Val Gly Asp Lys Thr Ala Trp Ile Leu Gly Ala Cys Lys Thr 85 90 95 Ser Ile Ser Arg Lys Gly Asn Met Thr Leu Ser Pro Glu Asn Gly Tyr 100 105 110 Trp Val Val Ile Met Met Lys Glu Asn Glu Tyr Gln Ala Ser Ser Val 115 120 125 Pro Pro Thr Arg Leu Leu Ile Lys Glu Pro Pro Lys Arg Val Gly Ile 130 135 140 Phe Val Asp Tyr Arg Val Gly Ser Ile Ser Phe Tyr Asn Val Thr Ala 145 150 155 160 Arg Ser His Ile Tyr Thr Phe Ala Ser Cys Ser Phe Ser Gly Pro Leu 165 170 175 Gln Pro Ile Phe Ser 180 6 20 DNA homo sapiens 6 aacctgcctt ttcttgctca 20 7 20 DNA homo sapiens 7 cactcagcac tggatgagga 20 8 26 DNA homo sapiens 8 atcattttgc atctggttgt ccttcc 26 9 26 DNA homo sapiens 9 tcccctgtag aaatggtgac ctcaag 26 10 26 DNA homo sapiens 10 ggccgggagg gggctgtcga ggaagc 26 11 26 DNA homo sapiens 11 tcgtgcccgg ccagccattc tttctc 26 12 22 DNA homo sapiens 12 tgagaactcg cacatctcag gc 22 13 22 DNA homo sapiens 13 aaggcccagt gtgtccaagt gc 22 14 21 DNA homo sapiens 14 ttggcaccag ctaaagatgg c 21 15 21 DNA homo sapiens 15 tctccctcta cagggatgag c 21 16 22 DNA homo sapiens 16 tatcgcctcc tgctctggaa tc 22 17 23 DNA homo sapiens 17 cactgtgggt caccaagacc aag 23 18 21 DNA homo sapiens 18 tccaggagcc cagaagtaga g 21 19 21 DNA homo sapiens 19 ttctccctat caaatccaga g 21 20 21 DNA homo sapiens 20 agaatgtagt tcatttccag c 21 21 20 DNA homo sapiens 21 catttctgaa cgcagggttt 20 22 23 DNA homo sapiens 22 acctaactcc agcttctctc tgc 23 23 21 DNA homo sapiens 23 agttcttctg gaacgtggta g 21 24 21 DNA homo sapiens 24 ccagaagaac taccctgtcc c 21 25 20 DNA homo sapiens 25 agagcagctg gcgaatgtat 20 26 20 DNA homo sapiens 26 gaggtggagg ttggagacaa 20 27 20 DNA homo sapiens 27 tcctcctctg aaatccatgg 20 28 20 DNA homo sapiens 28 aagctcactg ccttctcctc 20 29 20 DNA homo sapiens 29 gaggagaagg cagtgagctt 20 30 20 DNA homo sapiens 30 gacttggaaa caagtgggag 20 31 20 DNA homo sapiens 31 ctcccacttg tttccaagtc 20 32 17 DNA homo sapiens 32 gtaaaacgac ggccagt 17 33 20 DNA homo sapiens 33 caggaaacag ctatgaccat 20 34 18 DNA homo sapiens 34 gttttcccag tcacgacg 18 35 184 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 35 Val Asn Val Ile Leu Asp Ala Glu Thr Ala Tyr Pro Asn Leu Ile Phe 1 5 10 15 Ser Asp Asp Leu Lys Ser Val Arg Leu Gly Asn Lys Trp Glu Arg Leu 20 25 30 Pro Asp Gly Pro Gln Arg Phe Asp Ser Cys Ile Ile Val Leu Gly Ser 35 40 45 Pro Ser Phe Leu Ser Gly Arg Arg Tyr Trp Glu Val Glu Val Gly Asp 50 55 60 Lys Thr Ala Trp Ile Leu Gly Ala Cys Lys Thr Ser Ile Ser Arg Lys 65 70 75 80 Gly Asn Met Thr Leu Ser Pro Glu Asn Gly Tyr Trp Val Val Ile Met 85 90 95 Met Lys Glu Asn Glu Tyr Gln Ala Ser Ser Val Pro Pro Thr Arg Leu 100 105 110 Leu Ile Lys Glu Pro Pro Lys Arg Val Gly Ile Phe Val Asp Tyr Arg 115 120 125 Val Gly Ser Ile Ser Phe Tyr Met Val Thr Ala Arg Ser His Ile Tyr 130 135 140 Thr Phe Ala Ser Cys Ser Phe Ser Gly Pro Leu Gln Pro Ile Phe Ser 145 150 155 160 Pro Gly Thr Arg Asp Gly Gly Lys Asn Thr Ala Pro Leu Thr Ile Cys 165 170 175 Pro Val Gly Gly Gln Gly Pro Asp 180 36 183 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 36 Val Asp Val Thr Leu Asp Pro Asp Thr Ala Tyr Pro Ser Leu Ile Leu 1 5 10 15 Ser Asp Asn Leu Arg Gln Val Arg Tyr Ser Tyr Leu Gln Gln Asp Leu 20 25 30 Pro Asp Asn Pro Glu Arg Phe Asn Leu Phe Pro Cys Val Leu Gly Ser 35 40 45 Pro Cys Phe Ile Ala Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp 50 55 60 Lys Ala Lys Trp Thr Ile Gly Val Cys Glu Asp Ser Val Cys Arg Lys 65 70 75 80 Gly Gly Val Thr Ser Ala Pro Gln Asn Gly Phe Trp Ala Val Ser Leu 85 90 95 Trp Tyr Gly Lys Glu Tyr Trp Ala Leu Thr Ser Pro Met Thr Ala Leu 100 105 110 Pro Leu Arg Thr Pro Leu Gln Arg Val Gly Ile Phe Leu Asp Tyr Asp 115 120 125 Ala Gly Glu Val Ser Phe Tyr Asn Val Thr Glu Arg Cys His Thr Phe 130 135 140 Thr Phe Ser His Ala Thr Phe Cys Gly Pro Val Arg Pro Tyr Phe Ser 145 150 155 160 Leu Ser Tyr Ser Gly Gly Lys Ser Ala Ala Pro Leu Ile Ile Cys Pro 165 170 175 Met Ser Gly Ile Asp Gly Phe 180 37 178 PRT Xenopus Laevis 37 Thr Pro Met Leu Leu Asp Pro Thr Ser Ala His Pro Asn Leu His Leu 1 5 10 15 Ser Asp Gly Leu Thr Ser Val Arg Tyr Gly Glu Asn Lys Leu Ser Leu 20 25 30 Pro Asp Asn Pro Lys Ala Phe Ser Gln Cys Ile Leu Val Leu Gly Ser 35 40 45 Gln Gly Phe Asp Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp 50 55 60 Lys Thr Ala Trp Asp Val Gly Met Ala Ser Glu Ser Ser Asn Arg Lys 65 70 75 80 Gly Lys Ile Lys Leu Asn Pro Lys Asn Gly Tyr Trp Ala Ile Trp Leu 85 90 95 Arg Asn Gly Asn Ala Tyr Lys Ala Leu Glu Ser Pro Ser Lys Ser Leu 100 105 110 Ser Leu Ser Ser His Pro Arg Lys Ile Gly Val Tyr Val Asp Tyr Glu 115 120 125 Gly Gly Gln Ile Ser Phe Tyr Asn Ala Asp Asp Met Thr Ile Ile Tyr 130 135 140 Thr Phe Asn Ala Thr Phe Thr Glu Lys Leu Tyr Pro Tyr Leu Ser Pro 145 150 155 160 Phe Leu His Asp Ser Gly Lys Asn Val Asp Pro Leu Arg Phe Val His 165 170 175 Asn Lys 38 179 PRT Pleurodeles Waltl 38 Ala Pro Leu Thr Leu Asp Pro Asn Thr Ala His Pro Asn Leu Val Leu 1 5 10 15 Ser Glu Gly Leu Thr Ser Val Lys Tyr Thr Asp Thr Lys Gln Gln Leu 20 25 30 Pro Asp Asn Pro Lys Arg Phe Ser Gln Cys Ile Leu Val Leu Gly Ala 35 40 45 Glu Gly Phe Asp Ser Gly Lys His Tyr Trp Glu Val Glu Val Gly Asn 50 55 60 Lys Thr Ala Trp Asp Val Gly Met Ala Ser Glu Ser Ser Asn Arg Lys 65 70 75 80 Gly Lys Ile Lys Leu Asn Pro Lys Asn Gly Tyr Trp Ala Ile Trp Leu 85 90 95 Arg Asn Gly Asn Ala Phe Lys Ala Leu Glu Ser Pro Ser Lys Thr Leu 100 105 110 Asn Leu Thr Ser Lys Pro Ser Lys Ile Gly Val Tyr Leu Asp Tyr Glu 115 120 125 Gly Gly Gln Val Ser Phe Tyr Asn Ala Asp Asp Met Ser Pro Ile Tyr 130 135 140 Thr Phe Asn Gly Ser Phe Thr Glu Lys Leu Tyr Pro Tyr Leu Ser Pro 145 150 155 160 Phe Leu Gln Asp Ser Gly Lys Asn Ala Glu Pro Leu Lys Leu Val His 165 170 175 Thr Lys Leu 39 185 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 39 Val His Ile Thr Leu Asp Pro Asp Thr Ala Asn Pro Trp Leu Ile Leu 1 5 10 15 Ser Glu Asp Arg Arg Gln Val Arg Leu Gly Asp Thr Gln Gln Ser Ile 20 25 30 Pro Gly Asn Glu Glu Arg Phe Asp Ser Tyr Pro Met Val Leu Gly Ala 35 40 45 Gln His Phe His Ser Gly Lys His Tyr Trp Glu Val Asp Val Thr Gly 50 55 60 Lys Glu Ala Trp Asp Leu Gly Val Cys Arg Asp Ser Val Arg Arg Lys 65 70 75 80 Gly His Phe Leu Leu Ser Ser Lys Ser Gly Phe Trp Thr Ile Trp Leu 85 90 95 Trp Asn Lys Gln Lys Tyr Glu Ala Gly Thr Tyr Pro Gln Thr Pro Leu 100 105 110 His Leu Gln Val Pro Pro Cys Gln Val Gly Ile Phe Leu Asp Tyr Glu 115 120 125 Ala Gly Met Val Ser Phe Tyr Asn Ile Thr Asp His Gly Ser Leu Ile 130 135 140 Tyr Ser Phe Ser Glu Cys Ala Phe Thr Gly Pro Leu Arg Pro Phe Phe 145 150 155 160 Ser Pro Gly Phe Asn Asp Gly Gly Lys Asn Thr Ala Pro Leu Thr Leu 165 170 175 Cys Pro Leu Asn Ile Gly Ser Gln Gly 180 185 40 197 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 40 Val Ser Val Thr Leu Asp Pro Gln Ser Ala Ser Gly Tyr Leu Gln Leu 1 5 10 15 Ser Glu Asp Trp Lys Cys Val Thr Tyr Thr Ser Leu Tyr Lys Ser Ala 20 25 30 Tyr Leu His Pro Gln Gln Phe Asp Cys Glu Pro Gly Val Leu Gly Ser 35 40 45 Lys Gly Phe Thr Trp Gly Lys Val Tyr Trp Glu Val Glu Val Glu Arg 50 55 60 Glu Gly Trp Ser Glu Asp Glu Glu Glu Gly Asp Glu Glu Glu Glu Gly 65 70 75 80 Glu Glu Glu Glu Glu Glu Glu Glu Ala Gly Tyr Gly Asp Gly Tyr Asp 85 90 95 Asp Trp Glu Thr Asp Glu Asp Glu Glu Ser Leu Gly Asp Glu Glu Glu 100 105 110 Glu Glu Glu Glu Glu Glu Glu Glu Val Leu Glu Ser Cys Met Val Gly 115 120 125 Val Ala Arg Asp Ser Val Lys Arg Lys Gly Asp Leu Ser Leu Arg Pro 130 135 140 Glu Asp Gly Val Trp Ala Leu Arg Leu Ser Ser Ser Gly Ile Trp Ala 145 150 155 160 Asn Thr Ser Pro Glu Ala Glu Leu Phe Pro Ala Leu Arg Pro Arg Arg 165 170 175 Val Gly Ile Ala Leu Asp Tyr Glu Gly Gly Thr Val Thr Phe Thr Asn 180 185 190 Ala Glu Ser Gln Glu 195 41 174 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 41 Ala Asp Val Ile Leu Asp Pro Lys Thr Ala Asn Pro Ile Leu Leu Val 1 5 10 15 Ser Glu Asp Gln Arg Ser Val Gln Arg Ala Lys Glu Pro Gln Asp Leu 20 25 30 Pro Asp Asn Pro Glu Arg Phe Asn Trp His Tyr Cys Val Leu Gly Cys 35 40 45 Glu Ser Phe Ile Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp 50 55 60 Arg Lys Glu Trp His Ile Gly Val Cys Ser Lys Asn Val Gln Arg Lys 65 70 75 80 Gly Trp Val Lys Met Thr Pro Glu Asn Gly Phe Trp Thr Met Gly Leu 85 90 95 Thr Asp Gly Asn Lys Tyr Arg Thr Leu Thr Glu Pro Arg Thr Asn Leu 100 105 110 Lys Leu Pro Lys Pro Pro Lys Lys Val Gly Val Phe Leu Asp Tyr Glu 115 120 125 Thr Gly Asp Ile Ser Phe Tyr Asn Ala Val Asp Gly Ser His Ile His 130 135 140 Thr Phe Leu Asp Val Ser Phe Ser Glu Ala Leu Tyr Pro Val Phe Arg 145 150 155 160 Ile Leu Thr Leu Glu Pro Thr Ala Leu Ser Ile Cys Pro Ala 165 170 42 174 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 42 Ile Lys Val Ile Leu Asp Tyr Asn Thr Ala His Asn Lys Val Ala Leu 1 5 10 15 Ser Glu Cys Tyr Thr Val Ala Ser Val Ala Glu Met Pro Gln Asn Tyr 20 25 30 Arg Pro His Pro Gln Arg Phe Thr Tyr Cys Ser Gln Val Leu Gly Leu 35 40 45 His Cys Tyr Lys Lys Gly Ile His Tyr Trp Glu Val Glu Leu Gln Lys 50 55 60 Asn Asn Phe Cys Gly Val Gly Ile Cys Tyr Gly Ser Met Asn Arg Gln 65 70 75 80 Gly Pro Glu Ser Arg Leu Gly Arg Asn Ser Ala Ser Trp Cys Val Glu 85 90 95 Trp Phe Asn Thr Lys Ile Ser Ala Trp His Asn Asn Val Glu Lys Thr 100 105 110 Leu Pro Ser Thr Lys Ala Thr Arg Val Gly Val Leu Leu Asn Cys Asp 115 120 125 His Gly Phe Val Ile Phe Phe Ala Val Ala Asp Lys Val His Leu Met 130 135 140 Tyr Lys Phe Arg Val Asp Phe Thr Glu Ala Leu Tyr Pro Ala Phe Trp 145 150 155 160 Val Phe Ser Ala Gly Ala Thr Leu Ser Ile Cys Ser Pro Lys 165 170 43 164 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 43 His Ile Ser Leu Asp Pro Gln Thr Ser His Pro Lys Leu Leu Leu Ser 1 5 10 15 Lys Asp His Gln Arg Ala Gln Phe Ser Tyr Lys Trp Gln Asn Ser Pro 20 25 30 Asp Asn Pro Gln Arg Phe Asp Arg Ala Thr Cys Val Leu Ala His Thr 35 40 45 Gly Ile Thr Gly Gly Arg His Thr Trp Val Val Ser Ile Asp Leu Ala 50 55 60 His Gly Ala Ser Cys Thr Val Gly Val Val Ser Glu Asp Val Gln Arg 65 70 75 80 Lys Gly Glu Leu Arg Leu Arg Pro Glu Glu Gly Val Trp Ala Val Arg 85 90 95 Leu Ala Trp Gly Phe Val Ser Ala Leu Gly Ser Phe Pro Thr Arg Leu 100 105 110 Thr Leu Lys Glu Gln Pro Arg Gln Val Arg Val Ser Leu Asp Tyr Glu 115 120 125 Val Gly Trp Val Thr Phe Thr Asn Ala Val Thr Arg Glu Pro Ile Tyr 130 135 140 Thr Phe Thr Ala Ser Phe Thr Arg Lys Val Ile Pro Phe Phe Gly Leu 145 150 155 160 Trp Gly Arg Gly 44 144 PRT Porcine 44 Ala His Ile Ser Leu Asp Pro Gln Thr Ser His Pro Lys Leu Leu Leu 1 5 10 15 Ser Glu Asp Asn Gln Gln Ala Arg Phe Ser Tyr Lys Trp Gln Asn Ser 20 25 30 Pro Asp Asn Pro Gln Arg Phe Asp Arg Ala Thr Cys Val Leu Ala His 35 40 45 Ser Gly Phe Thr Glu Gly Arg His Thr Trp Val Val Ser Val Asp Leu 50 55 60 Ala His Gly Gly Ser Cys Thr Val Gly Val Val Ser Gln Asp Ile Arg 65 70 75 80 Arg Lys Gly Glu Leu Arg Met Arg Pro Glu Glu Gly Val Trp Ala Val 85 90 95 Arg Leu Ala Trp Gly Phe Val Ser Ala Leu Gly Ser Phe Pro Thr Arg 100 105 110 Leu Ala Leu Glu Glu His Pro Arg Gln Val Arg Val Ser Ile Asp Tyr 115 120 125 Glu Val Gly Trp Val Thr Phe Val Asn Ala Val Thr Gln Glu Pro Ile 130 135 140 45 146 PRT Artificial Sequence Description of Artificial Sequence/ Note = Synthetic construct 45 Val Asp Val Met Leu Asn Pro Gly Ser Ala Thr Ser Asn Val Ala Ile 1 5 10 15 Ser Val Asp Gln Arg Gln Val Lys Thr Val Arg Thr Cys Thr Phe Lys 20 25 30 Asn Ser Asn Pro Cys Asp Phe Ser Ala Phe Gly Val Phe Gly Cys Gln 35 40 45 Tyr Phe Ser Ser Gly Lys Tyr Tyr Trp Glu Val Asp Val Ser Gly Lys 50 55 60 Ile Ala Trp Ile Leu Gly Val His Ser Lys Ile Ser Ser Leu Asn Lys 65 70 75 80 Arg Lys Ser Ser Gly Phe Ala Phe Asp Pro Ser Val Asn Tyr Ser Lys 85 90 95 Val Tyr Ser Arg Tyr Arg Pro Gln Tyr Gly Tyr Trp Val Ile Gly Leu 100 105 110 Gln Asn Thr Cys Glu Tyr Asn Ala Phe Glu Asp Ser Ser Ser Ser Asp 115 120 125 Pro Lys Val Leu Thr Leu Phe Met Ala Val Leu Pro Val Val Leu Gly 130 135 140 Phe Ser 145 

What is claimed is:
 1. An isolated nucleic acid sequence, comprising the coding sequence of SEQ ID NO: 2 or a nucleic acid encoding SEQ ID NO:
 3. 2. An isolated nucleic acid sequence consisting of the sequence of SEQ ID NO:
 1. 3. An isolated nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 and SEQ ID NO:12. 